Coronavirus vaccine compositions and methods

ABSTRACT

Provided herein are nucleic acid molecules encoding viral replication proteins and antigenic coronavirus proteins or fragments thereof. Also provided herein are compositions that include nucleic acid molecules encoding viral replication and antigenic proteins, and lipids. Nucleic acid molecules provided herein are useful for inducing immune responses.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/987,191, filed Mar. 9, 2020 and U.S. Provisional Application No.63/073,900, filed Sep. 2, 2020.

REFERENCE TO A SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 8, 2021 isnamed 049386-538001US_SequenceListing_ST25.txt and is 481,150 bytes insize.

TECHNICAL FIELD

The present disclosure relates generally to inducing immune responsesagainst infectious agents and tumor antigens and more specifically toself-transcribing and replicating RNA for antigen expression.

BACKGROUND

Infectious diseases and cancer represent significant burdens on healthworldwide. According to the World Health Organization (WHO), lowerrespiratory tract infection was the deadliest infectious diseaseworldwide in 2016, causing approximately 3 million deaths. The impact ofinfectious diseases is illustrated by the coronavirus disease 2019(COVID-19) pandemic caused by severe acute respiratorysyndrome-coronavirus-2 (SARS-CoV-2). SARS-CoV-2 is a novel coronavirusthat was first identified in December 2019 in Wuhan, China and that hascaused more than 20 million confirmed infections with more than 700,000deaths worldwide as of August 2020. Current control measures to curb therapid worldwide spread of SARS-CoV-2, such as national lockdowns,closure of work places and schools, and reduction of internationaltravel are threatening to result in a global economic recession to anextent not seen since the Great Depression.

Cancer is the second leading cause of death globally, accounting forapproximately 9.6 million deaths worldwide in 2018. Cancer is a largegroup of diseases that can affect almost any organ or tissue in thebody. Cancer burden continues to grow globally, exerting physical,emotional, and financial strains on patients and health care providers.

Self-replicating ribonucleic acids (RNAs), e.g., derived from viralreplicons, are useful for expression of proteins, such as heterologousproteins, for a variety of purposes, such as expression of therapeuticproteins and expression of antigens for vaccines. A desirable propertyof such replicons is the ability for sustained expression of theprotein.

Few treatments for infections caused by viruses and eukaryotic organismsare available, and resistance to antibiotics for the treatment ofbacterial infections is increasing. In addition, rapid responses,including rapid vaccine development, are required to effectively controlemerging infectious diseases and pandemics. Moreover, many cancertreatments include costly and painful surgeries and chemotherapies thatare often unsuccessful or only modestly prolong life despite seriousside effects. Thus, there exists a need for the prevention and/ortreatment of infectious diseases and cancer.

SUMMARY

In one aspect, the present disclosure provides a nucleic acid moleculecomprising (i) a first polynucleotide encoding one or more viralreplication proteins, wherein the first polynucleotide iscodon-optimized as compared to a wild-type polynucleotide encoding theone or more viral replication proteins; and (ii) a second polynucleotidecomprising a first transgene encoding a first antigenic protein or afragment thereof, wherein the first antigenic protein is a coronavirusprotein.

In some embodiments, the one or more viral replication proteins arealphavirus proteins or rubivirus proteins.

In some embodiments, the alphavirus proteins are from Venezuelan EquineEncephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV),Everglades Virus (EVEV), Mucambo Virus (MUCV), Semliki Forest Virus(SFV), Pixuna Virus (PIXV), Middleburg Virus (MIDV), Chikungunya Virus(CHIKV), O'Nyong-Nyong Virus (ONNV), Ross River Virus (RRV), BarmahForest Virus (BFV), Getah Virus (GETV), Sagiyama Virus (SAGV), BebaruVirus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), Sindbis Virus(SINV), Aura Virus (AURAV), Whataroa Virus (WHAV), Babanki Virus (BABV),Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus (WEEV),Highland J Virus (HJV), Fort Morgan Virus (FMV), Ndumu Virus (NDUV),Salmonid Alphavirus (SAV), Buggy Creek Virus (BCRV), or any combinationthereof.

In some embodiments, the first polynucleotide encodes a polyproteincomprising an alphavirus nsP1 protein, an alphavirus nsP2 protein, analphavirus nsP3 protein, an alphavirus nsP4 protein, or any combinationthereof.

In some embodiments, the first polynucleotide encodes a polyproteincomprising an alphavirus nsP1 protein, an alphavirus nsP2 protein, analphavirus nsP3 protein, or any combination thereof, and an alphavirusnsP4 protein.

In some embodiments, the nucleic acid molecule further comprises a firstintergenic region between a sequence encoding the polyprotein comprisingan alphavirus nsP1 protein, an alphavirus nsP2 protein, an alphavirusnsP3 protein, or any combination thereof, and a sequence encoding analphavirus nsP4 protein.

In some embodiments, the first intergenic region comprises an alphavirussequence.

In some embodiments, the first polynucleotide comprises a sequencehaving at least 80% identity to a sequence of SEQ ID NO:72.

In some embodiments, the nucleic acid molecule further comprises a 5′untranslated region (UTR).

In some embodiments, the 5′ UTR comprises a viral 5′ UTR, a non-viral 5′UTR, or a combination of viral and non-viral 5′ UTR sequences.

In some embodiments, the 5′ UTR comprises an alphavirus 5′ UTR.

In some embodiments, the alphavirus 5′ UTR comprises a Venezuelan EquineEncephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV),Everglades Virus (EVEV), Mucambo Virus (MUCV), Semliki Forest Virus(SFV), Pixuna Virus (PIXV), Middleburg Virus (MIDV), Chikungunya Virus(CHIKV), O'Nyong-Nyong Virus (ONNV), Ross River Virus (RRV), BarmahForest Virus (BFV), Getah Virus (GETV), Sagiyama Virus (SAGV), BebaruVirus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), Sindbis Virus(SINV), Aura Virus (AURAV), Whataroa Virus (WHAV), Babanki Virus (BABV),Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus (WEEV),Highland J Virus (HJV), Fort Morgan Virus (FMV), Ndumu Virus (NDUV),Salmonid Alphavirus (SAV), or Buggy Creek Virus (BCRV) 5′ UTR sequence.

In some embodiments, the 5′ UTR comprises a sequence of SEQ ID NO:73,SEQ ID NO:74, or SEQ ID NO:75.

In some embodiments, the nucleic acid molecule further comprises a 3′untranslated region (UTR).

In some embodiments, the 3′ UTR comprises a viral 3′ UTR, a non-viral 3′UTR, or a combination of viral and non-viral 3′ UTR sequences. In someembodiments, the 3′ UTR comprises an alphavirus 3′ UTR.

In some embodiments, the alphavirus 3′ UTR comprises a Venezuelan EquineEncephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV),Everglades Virus (EVEV), Mucambo Virus (MUCV), Semliki Forest Virus(SFV), Pixuna Virus (PIXV), Middleburg Virus (MIDV), Chikungunya Virus(CHIKV), O'Nyong-Nyong Virus (ONNV), Ross River Virus (RRV), BarmahForest Virus (BFV), Getah Virus (GETV), Sagiyama Virus (SAGV), BebaruVirus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), Sindbis Virus(SINV), Aura Virus (AURAV), Whataroa Virus (WHAV), Babanki Virus (BABV),Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus (WEEV),Highland J Virus (HJV), Fort Morgan Virus (FMV), Ndumu Virus (NDUV),Salmonid Alphavirus (SAV), or Buggy Creek Virus (BCRV) 3′ UTR sequence.

In some embodiments, the 3′ UTR comprises a poly-A sequence.

In some embodiments, the 3′ UTR comprises a sequence of SEQ ID NO:76.

In some embodiments, the antigenic protein is a SARS-CoV-2 protein.

In some embodiments, the antigenic protein is a SARS-CoV-2 spikeglycoprotein.

In some embodiments, the SARS-CoV-2 spike glycoprotein is a wild-typeSARS-CoV-2 spike glycoprotein having an amino acid sequence of SEQ IDNO:123.

In some embodiments, the second polynucleotide comprises a sequencehaving at least 85% identity to a sequence of SEQ ID NO:121 or SEQ IDNO:122.

In some embodiments, the second polynucleotide comprises at least twotransgenes.

In some embodiments, a second transgene encodes a second antigenicprotein or a fragment thereof or an immunomodulatory protein.

In some embodiments, the second polynucleotide further comprises asequence encoding a 2A peptide, an internal ribosomal entry site (IRES),or a combination thereof, located between transgenes.

In some embodiments, the immunomodulatory protein is a cytokine, achemokine, or an interleukin.

In some embodiments, the second transgene encodes a second coronavirusprotein.

In some embodiments, the first polynucleotide is located 5′ of thesecond polynucleotide.

In some embodiments, the nucleic acid molecule further comprises asecond intergenic region located between the first polynucleotide andthe second polynucleotide.

In some embodiments, the second intergenic region comprises a sequencehaving at least 85% identity to a sequence of SEQ ID NO:77.

In some embodiments, the nucleic acid molecule is

-   -   (a) a DNA molecule; or    -   (b) an RNA molecule, wherein T is substituted with U.

In some embodiments, the DNA molecule further comprises a promoter.

In some embodiments, the promoter is located 5′ of the 5′UTR.

In some embodiments, the promoter is a T7 promoter, a T3 promoter, or anSP6 promoter.

In some embodiments, the RNA molecule is a self-replicating RNAmolecule.

In some embodiments, the RNA molecule further comprises a 5′ cap.

In some embodiments, the 5′ cap has a Cap 1 structure, a Cap 1 (^(m6)A)structure, a Cap 2 structure, a Cap 0 structure, or any combinationthereof.

In another aspect, the disclosure provides a nucleic acid moleculecomprising

-   -   (a) a sequence of SEQ ID NO:124;    -   (b) a sequence of SEQ ID NO:124, wherein T is substituted with        U;    -   (c) a sequence of SEQ ID NO:125; or    -   (d) a sequence of SEQ ID NO:125, wherein T is substituted with        U.

In some embodiments, the nucleic acid molecule is an RNA molecule.

In some embodiments, the nucleic acid molecule further comprises a 5′cap having a Cap 1 structure.

In yet another aspect the disclosure provides a nucleic acid moleculecomprising:

-   -   (i) a first polynucleotide comprising a sequence having at least        80% identity to a sequence of SEQ ID NO:72; and    -   (ii) a second polynucleotide comprising a first transgene        encoding a first antigenic protein or a fragment thereof,        wherein the first antigenic protein is a coronavirus protein.

In some embodiments, the nucleic acid molecule further comprises a 5′untranslated region (UTR).

In some embodiments, the 5′ UTR comprises a viral 5′ UTR, a non-viral 5′UTR, or a combination of viral and non-viral 5′ UTR sequences.

In some embodiments, the 5′ UTR comprises an alphavirus 5′ UTR.

In some embodiments, the alphavirus 5′ UTR comprises a Venezuelan EquineEncephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV),Everglades Virus (EVEV), Mucambo Virus (MUCV), Semliki Forest Virus(SFV), Pixuna Virus (PIXV), Middleburg Virus (MIDV), Chikungunya Virus(CHIKV), O'Nyong-Nyong Virus (ONNV), Ross River Virus (RRV), BarmahForest Virus (BFV), Getah Virus (GETV), Sagiyama Virus (SAGV), BebaruVirus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), Sindbis Virus(SINV), Aura Virus (AURAV), Whataroa Virus (WHAV), Babanki Virus (BABV),Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus (WEEV),Highland J Virus (HJV), Fort Morgan Virus (FMV), Ndumu Virus (NDUV),Salmonid Alphavirus (SAV), or Buggy Creek Virus (BCRV) 5′ UTR sequence.

In some embodiments, the 5′ UTR comprises a sequence of SEQ ID NO:73,SEQ ID NO:74, or SEQ ID NO:75.

In some embodiments, the nucleic acid molecule further comprises a 3′untranslated region (UTR).

In some embodiments, the 3′ UTR comprises a viral 3′ UTR, a non-viral 3′UTR, or a combination of viral and non-viral 3′ UTR sequences.

In some embodiments, the 3′ UTR comprises an alphavirus 3′ UTR.

In some embodiments, the alphavirus 3′ UTR comprises a Venezuelan EquineEncephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV),Everglades Virus (EVEV), Mucambo Virus (MUCV), Semliki Forest Virus(SFV), Pixuna Virus (PIXV), Middleburg Virus (MIDV), Chikungunya Virus(CHIKV), O'Nyong-Nyong Virus (ONNV), Ross River Virus (RRV), BarmahForest Virus (BFV), Getah Virus (GETV), Sagiyama Virus (SAGV), BebaruVirus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), Sindbis Virus(SINV), Aura Virus (AURAV), Whataroa Virus (WHAV), Babanki Virus (BABV),Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus (WEEV),Highland J Virus (HJV), Fort Morgan Virus (FMV), Ndumu Virus (NDUV),Salmonid Alphavirus (SAV), or Buggy Creek Virus (BCRV) 3′ UTR sequence.

In some embodiments, the 3′ UTR comprises a poly-A sequence.

In some embodiments, the 3′ UTR comprises a sequence of SEQ ID NO:76.

In some embodiments, the antigenic protein is a SARS-CoV-2 protein.

In some embodiments, the antigenic protein is a SARS-CoV-2 spikeglycoprotein.

In some embodiments, the SARS-CoV-2 spike glycoprotein is a wild-typeSARS-CoV-2 spike glycoprotein having an amino acid sequence of SEQ IDNO:123.

In some embodiments, the second polynucleotide comprises a sequencehaving at least 85% identity to a sequence of SEQ ID NO:121 or SEQ IDNO:122.

In some embodiments, the second polynucleotide comprises at least twotransgenes.

In some embodiments, a second transgene encodes a second antigenicprotein or a fragment thereof or an immunomodulatory protein.

In some embodiments, the second polynucleotide further comprises asequence encoding a 2A peptide, an internal ribosomal entry site (IRES),or a combination thereof, located between transgenes.

In some embodiments, the immunomodulatory protein is a cytokine, achemokine, or an interleukin.

In some embodiments, the second transgene encodes a second coronavirusprotein.

In some embodiments, the first polynucleotide is located 5′ of thesecond polynucleotide.

In some embodiments, the nucleic acid molecule further comprises asecond intergenic region located between the first polynucleotide andthe second polynucleotide.

In some embodiments, the second intergenic region comprises a sequencehaving at least 85% identity to a sequence of SEQ ID NO:77.

In some embodiments, the nucleic acid molecule is

-   -   (a) a DNA molecule; or    -   (b) an RNA molecule, wherein T is substituted with U.

In some embodiments, the DNA molecule further comprises a promoter.

In some embodiments, the promoter is located 5′ of the 5′UTR.

In some embodiments, the promoter is a T7 promoter, a T3 promoter, or anSP6 promoter.

In some embodiments, the RNA molecule is a self-replicating RNAmolecule.

In some embodiments, the RNA molecule further comprises a 5′ cap.

In some embodiments, the 5′ cap has a Cap 1 structure, a Cap 1 (^(m6)A)structure, a Cap 2 structure, a Cap 0 structure, or any combinationthereof.

In yet another aspect, the disclosure provides a composition comprisingany of the nucleic acid molecules provided herein. In some embodiments,the composition further comprises a lipid.

In some embodiments, the lipid comprises an ionizable cationic lipid.

In some embodiments, the ionizable cationic lipid has a structure of

or a pharmaceutically acceptable salt thereof.

In yet another aspect, the disclosure provides a composition comprisingany of the nucleic acid molecules described herein and a lipidformulation.

In some embodiments, the lipid formulation comprises an ionizablecationic lipid.

In some embodiments, the ionizable cationic lipid has a structure of

or a pharmaceutically acceptable salt thereof.

In some embodiments, the lipid formulation is selected from a lipoplex,a liposome, a lipid nanoparticle, a polymer-based carrier, an exosome, alamellar body, a micelle, and an emulsion.

In some embodiments, the lipid formulation is a liposome selected from acationic liposome, a nanoliposome, a proteoliposome, a unilamellarliposome, a multilamellar liposome, a ceramide-containing nanoliposome,and a multivesicular liposome.

In some embodiments, the lipid formulation is a lipid nanoparticle.

In some embodiments, the lipid nanoparticle has a size of less thanabout 200 nm. In some embodiments, the lipid nanoparticle has a size ofless than about 150 nm. In some embodiments, the lipid nanoparticle hasa size of less than about 100 nm. In some embodiments, the lipidnanoparticle has a size of about 55 nm to about 90 nm.

In some embodiments, the lipid formulation comprises one or morecationic lipids.

In some embodiments, the one or more cationic lipids is selected from5-carboxyspermylglycinedioctadecylamide (DOGS),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium(DOSPA), 1,2-Dioleoyl-3-Dimethylammonium-Propane (DODAP),1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP),1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA),N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane(CLinDMA), 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy1-1-(cis,cis-9′,1-2′-octadecadienoxy)propane (CpLinDMA),N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP),1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), and2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or(DLin-K-XTC2-DMA).

In some embodiments, the lipid formulation comprises an ionizablecationic lipid.

In some embodiments, the ionizable cationic lipid has a structure ofFormula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein R⁵ andR⁶ are each independently selected from the group consisting of a linearor branched C₁-C₃₁ alkyl, C₂-C₃₁ alkenyl or C₂-C₃₁ alkynyl andcholesteryl; L⁵ and L⁶ are each independently selected from the groupconsisting of a linear C₁-C₂₀ alkyl and C₂-C₂₀ alkenyl; X⁵ is —C(O)O—,whereby —C(O)O—R⁶ is formed or —OC(O)— whereby —OC(O)—R⁶ is formed; X⁶is —C(O)O— whereby —C(O)O—R⁵ is formed or —OC(O)— whereby —OC(O)—R⁵ isformed; X⁷ is S or O; L is absent or lower alkyl; R⁴ is a linear orbranched C₁-C₆ alkyl; and R⁷ and R⁸ are each independently selected fromthe group consisting of a hydrogen and a linear or branched C₁-C₆ alkyl.

In some embodiments, the ionizable cationic lipid is selected from

In some embodiments, the ionizable cationic lipid is ATX-126:

In some embodiments, the lipid formulation encapsulates the nucleic acidmolecule.

In some embodiments, the lipid formulation is complexed to the nucleicacid molecule.

In some embodiments, the lipid formulation further comprises a helperlipid. In some embodiments, the helper lipid is a phospholipid. In someembodiments, the helper lipid is selected from diolcoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC),distearoylphosphatidyl choline (DSPC), dimyristoylphosphatidyl glycerol(DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine(PC). In specific embodiments, the helper lipid isdistearoylphosphatidylcholine (DSPC).

In some embodiments, the lipid formulation further comprisescholesterol.

In some embodiments, the lipid formulation further comprises apolyethylene glycol (PEG)-lipid conjugate. In some embodiments, thePEG-lipid conjugate is PEG-DMG. In some embodiments, the PEG-DMG isPEG2000-DMG.

In some embodiments, the lipid portion of the lipid formulationcomprises about 40 mol % to about 60 mol % of the ionizable cationiclipid, about 4 mol % to about 16 mol % DSPC, about 30 mol % to about 47mol % cholesterol, and about 0.5 mol % to about 3 mol % PEG2000-DMG.

In some embodiments, the lipid portion of the lipid formulationcomprises about 42 mol % to about 58 mol % of the ionizable cationiclipid, about 6 mol % to about 14 mol % DSPC, about 32 mol % to about 44mol % cholesterol, and about 1 mol % to about 2 mol % PEG2000-DMG.

In some embodiments, the lipid portion of the lipid formulationcomprises about 45 mol % to about 55 mol % of the ionizable cationiclipid, about 8 mol % to about 12 mol % DSPC, about 35 mol % to about 42mol % cholesterol, and about 1.25 mol % to about 1.75 mol % PEG2000-DMG.

In some embodiments, the composition has a total lipid:nucleic acidmolecule weight ratio of about 50:1 to about 10:1. In some embodiments,the composition has a total lipid:nucleic acid molecule weight ratio ofabout 44:1 to about 24:1. In some embodiments, the composition has atotal lipid:nucleic acid molecule weight ratio of about 40:1 to about28:1. In some embodiments, the composition has a total lipid:nucleicacid molecule weight ratio of about 38:1 to about 30:1. In someembodiments, the composition has a total lipid:nucleic acid moleculeweight ratio of about 37:1 to about 33:1. In some embodiments, thecomposition comprises a HEPES or TRIS buffer at a pH of about 7.0 toabout 8.5.

In some embodiments, the HEPES or TRIS buffer is at a concentration ofabout 7 mg/mL to about 15 mg/mL.

In some embodiments, the composition further comprises about 2.0 mg/mLto about 4.0 mg/mL of NaCl.

In some embodiments, the composition further comprises one or morecryoprotectants.

In some embodiments, the one or more cryoprotectants are selected fromsucrose, glycerol, or a combination of sucrose and glycerol.

In some embodiments, the composition comprises a combination of sucroseat a concentration of about 70 mg/mL to about 110 mg/mL of sucrose andglycerol at a concentration of about 50 mg/mL to about 70 mg/mL.

In some embodiments, the composition is a lyophilized composition.

In some embodiments, the lyophilized composition comprises one or morelyoprotectants.

In some embodiments, the lyophilized composition comprises a poloxamer,potassium sorbate, sucrose, or any combination thereof.

In some embodiments, the poloxamer is poloxamer 188.

In some embodiments, the lyophilized composition comprises about 0.01 toabout 1.0% w/w of the nucleic acid molecule.

In some embodiments, the lyophilized composition comprises about 1.0 toabout 5.0% w/w lipids.

In some embodiments, the lyophilized composition comprises about 0.5 toabout 2.5% w/w of TRIS buffer.

In some embodiments, the lyophilized composition comprises about 0.75 toabout 2.75% w/w of NaCl.

In some embodiments, the lyophilized composition comprises about 85 toabout 95% w/w of a sugar. In some embodiments, the sugar is sucrose.

In some embodiments, the lyophilized composition comprises about 0.01 toabout 1.0% w/w of a poloxamer. In some embodiments, the poloxamer ispoloxamer 188.

In some embodiments, the lyophilized composition comprises about 1.0 toabout 5.0% w/w of potassium sorbate.

In some embodiments, the nucleic acid molecule comprises

-   -   (a) a sequence of SEQ ID NO:124;    -   (b) a sequence of SEQ ID NO:124, wherein T is substituted with        U;    -   (c) a sequence of SEQ ID NO:125; or    -   (d) a sequence of SEQ ID NO:125, wherein T is substituted with        U.

In yet another aspect, the disclosure provides a lipid nanoparticlecomposition comprising

-   -   a. a lipid formulation comprising        -   i. about 45 mol % to about 55 mol % of an ionizable cationic            lipid having the structure of ATX-126:

-   -   -   ii. about 8 mol % to about 12 mol % DSPC;        -   iii. about 35 mol % to about 42 mol % cholesterol; and        -   iv. about 1.25 mol % to about 1.75 mol % PEG2000-DMG; and

    -   b. a nucleic acid molecule having at least 85% sequence identity        to SEQ ID NO:125;        wherein the lipid formulation encapsulates the nucleic acid        molecule and the lipid nanoparticle has a size of about 60 to        about 90 nm.

In yet another aspect, the disclosure provides a method foradministering any of the compositions described herein to a subject inneed thereof, wherein the composition is administered intramuscularly,subcutaneously, intradermally, transdermally, intranasally, orally,sublingually, intravenously, intraperitoneally, topically, by aerosol,or by a pulmonary route. In specific embodiments, n the composition isadministered intramuscularly.

In yet another aspect, the disclosure provides a method of administeringany of the compositions described herein to a subject in need thereof,wherein the composition is lyophilized and is reconstituted prior toadministration.

In yet another aspect, the disclosure provides a method of amelioratingCOVID-19, comprising administering any of the compositions describedherein to a subject in need thereof.

In some embodiments, the composition is administered one time. In someembodiments, the composition is administered two times.

In yet another aspect, the disclosure provides a method of administeringa booster dose to a vaccinated subject, comprising administering any ofthe compositions described herein to a subject who was previouslyvaccinated against coronavirus.

In some embodiments, the composition is administered at a dosage ofabout 0.01 μg to about 1,000 μg of nucleic acid.

In some embodiments, the composition is administered at a dosage ofabout 1, 2, 5, 7.5, or 10 μg of nucleic acid.

In yet another aspect, the disclosure provides a method of inducing animmune response in a subject comprising administering to the subject aneffective amount of any of the nucleic acid molecules described herein.

In some embodiments, the nucleic acid molecule may be administeredintramuscularly, subcutaneously, intradermally, transdermally,intranasally, orally, sublingually, intravenously, intraperitoneally,topically, by aerosol, or by a pulmonary route.

In yet another aspect, the disclosure provides a method of inducing animmune response in a subject comprising administering to the subject aneffective amount of any of the compositions described herein.

In some embodiments, the composition may be administeredintramuscularly, subcutaneously, intradermally, transdermally,intranasally, orally, sublingually, intravenously, intraperitoneally,topically, by aerosol, or by a pulmonary route.

In some embodiments, the nucleic acid molecules described herein may beused in inducing an immune response to the first antigenic protein orfragment thereof.

In some embodiments, the nucleic acid molecules described herein may beused in the manufacture of a medicament for inducing an immune responseto the first antigenic protein or fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show design and expression of a SARS-CoV-2 vaccine in mRNAand self-replicating RNA (STARR™) platforms. (1A) Schematic diagram ofthe SARS-CoV-2 self-replicating STARR™ RNA and mRNA vaccine constructs.The STARR™ construct encodes for the four non-structural proteins,ns1-ns4, from Venezuelan equine encephalitis virus (VEEV) and theSARS-CoV-2 full length spike (S) protein. The mRNA construct codes forthe SARS-CoV-2 full length spike S protein. (1B) Physicalcharacteristics and RNA trapping efficiency of the LNP in the mRNA andSTARR™ (self-replicating RNA corresponding to SEQ ID NO:125; referred toherein as “STARR™ SARS-CoV-2 RNA”) vaccines. (1C) Western blot detectionof SARS-CoV-2 S protein following transfection of HEK293 cells with theSTARR™ RNA and mRNA constructs. (1D) In vivo comparison of proteinexpression following intramuscular (IM) administration of LNP containingluciferase-expressing STARR™ RNA or mRNA. Balb/c mice (n=3/group) wereinjected IM with 0.2 μg, 2.0 μg and 10.0 μg of STARR™ RNA or mRNA inlipid formulation. Luciferase expression was measured by in vivobioluminescence on days 1, 3 and 7 post-IM administration. S domain1=S1, S domain 2=S2, transmembrane domain=TM, cytoplasmic domain=CP.

FIGS. 2A-2I show clinical scores, mouse weights and transcriptomicanalysis of immune genes following vaccination with STARR™ RNA or mRNASARS-CoV-2 vaccine candidates. (2A) C57BL/6 mice were immunized witheither PBS, mRNA or STARR™ SARS-CoV-2 RNA (doses 0.2 μg, 2 μg or 10 μg),weight and clinical scores assessed every day, bled at day 1post-immunization, sacrificed at 7 days post-vaccination and lymph nodesharvested. Gene expression of inflammatory genes and immune genes weremeasured in whole blood (at day 1) and lymph nodes (at day 7),respectively. (2B) Expression of IFN and inflammatory response genes inwhole blood presented as heatmap of z scores. (2C) Lymph node weights at7 days post-vaccination. Principal component analysis (PCA) of immunegene expression following vaccination with mRNA or STARR™ SARS-CoV-2 RNAat doses (2D) 0.2 μg, (2E) 2 μg and (2F) 10 μg. Volcano plots of foldchange of STARR™ SARS-CoV-2 RNA versus mRNA (x-axis) and Log 10 P-valueof STARR™ SARS-CoV-2 RNA versus mRNA (y-axis) for doses (2G) 0.2 μg,(2H) 2 μg and (2I) 10 μg.

FIGS. 3A-3J show cellular immune responses following vaccination withSARS-CoV-2 STARR™ RNA and mRNA. C57BL/6 mice (n=5 per group) wereimmunized with 0.2 μg, 2 μg, or 10 μg of STARR™ RNA or mRNA via IM,sacrificed at day 7 post-vaccination and spleens analyzed for cellular Tcell responses by flow-cytometry and ELISPOT. (3A-3B) CD8+ and C) CD4+ Teffector cells were assessed in vaccinated animals using surfacestaining for T cell markers and flow-cytometry. (3D-3E) IFNγ+ CD8+ Tcells and (3F) Ratio of IFNγ+/IL4+CD4+ T cells in spleens of immunizedmice were assessed following ex vivo stimulation with PMA/ionomycin (IO)and intracellular staining. (3G-3I) SARS-CoV-2 S protein-specificresponses to pooled S protein peptides were assessed using IFNγ ELISPOTassays following vaccination with mRNA (3H) or STARR™ RNA (3I). Aschematic of S protein domains is shown in (3J).

FIGS. 4A-4G show humoral responses in multiple mouse strains followingimmunization with mRNA and STARR™ vaccine candidates. (4A) BALB/c andC57BL/6J mice were immunized via IM with 0.2 μg, 2 μg, or 10 Hg ofSTARR™ RNA or mRNA (n=5/group). Blood sampling was conducted atbaseline, and days 10, 19, 30, 40, 50 and 60 post-vaccination for BALB/cand days 10, 20 and 30 for C57BL/6J. (4B-4C) IgM and (4D-4E) IgG againstthe SARS-CoV-2 S protein over time, assessed using insect cell-derivedwhole S protein in a Luminex immuno-assay (measured as MFI). IgGendpoint titers to mammalian-derived whole S protein, S1, S2 andreceptor binding domain (RBD) proteins at day 30 post-vaccination wereassessed in (4F) BALB/c and (4G) C57BL/6J.

FIGS. 5A-5D show that STARR™ SARS-CoV-2 RNA elicits Th1 skewed immuneresponses. SARS-CoV-2 spike-specific IgG subclasses and the ratio ofIgG2a/c/IgG1 at 30 days post-vaccination with STARR™ RNA and mRNA in(5A) BALB/c and (5B) C57BL/6J mice. Th2 cytokine and Th1/Th2 skew in CD4T cells at day 7 post-vaccination in C57BL/6J mice measured by ICS as(5C) percentage of IL4+CD4 T cells and (5D) ratio of IFNγ+/IL4+ CD4+ Tcells.

FIGS. 6A-6E show that STARR™ SARS-CoV-2 RNA elicits a higher qualityhumoral response than mRNA platform. (6A) Avidity of SARS-CoV-2 Sprotein-specific IgG at day 30 post-immunization was measured using 8Murea washes. (6B) Neutralizing antibody (PRNT50 titers) at day 30post-vaccination against a clinically isolated live SARS-CoV-2 virusmeasured in both BALB/c and C57BL/6J. Dashed lines depict the serumdilution range (i.e. from 1:20 to 1:320) tested by PRNT. (6C) PRNT50 and(6D) PRNT70 of SARS-CoV-2 neutralization at day 60 post-vaccination andconvalescent sera from COVID-19 patients. (6E) Correlation analysis ofSpike-specific IgG endpoint titers against SARS-CoV-2 neutralization(PRNT50). PRNT—plaque reduction neutralization test.

FIGS. 7A-7E show clinical scores, body weight and immune responses toSTARR™ SARS-CoV-2 RNA and mRNA following boost at day 30 post-prime inC57BL/6J. (7A) Clinical scores and (7B) percentage of initial bodyweight following boost vaccinations. (7C) Anti-Spike IgG responsesfollowing boost by mRNA and STARR™ SARS-CoV-2 RNA. Grey dashed linemarks the experimental assay saturation point. IFN γ+ CD8+ T effectorcells responses (fold change over PBS) in animals either primed or prime& boosted with either (7D) mRNA or (7E) STARR™ SARS-CoV-2 RNA vaccinecandidates.

FIGS. 8A-8B show whole blood transcriptomic data at 1-day post-primevaccination showing Nanostring counts per 50 ng RNA of selected (8A) IFNand (8B) inflammatory genes.

FIGS. 9A-9B show correlation analysis of live SARS-CoV-2 neutralizationagainst binding IgG and IgG subclasses in BALB/c and C57BL/6J mousestrains. (9A) Spearman correlation analysis of SARS-CoV-2 neutralization(PRNT50) against total IgG specific to several SARS-CoV-2 antigens,including S, S1, and RBD recombinant proteins. (9B) Spearman correlationanalysis of SARS-CoV-2 neutralization (PRNT50) against SARS-CoV-2S-specific IgG subclasses (IgG1 and IgG2a or IgG2c).

FIG. 10 shows Kaplan-Meier survival curves for unvaccinated mice (PBS)and mice vaccinated with STARR™ SARS-CoV-2 RNA following challenge witha lethal dose of SARS-CoV-2 virus. Upper line—STARR™ SARS-CoV-2 RNA (2μg, 0 μg); dropping line—PBS.

FIG. 11 shows that STARR™ SARS-CoV-2 RNA vaccination protects againstlung and brain SARS-CoV-2 infection. Viral RNA levels in lungs (FIG. 11, left) and in brains (FIG. 11 , right) of unvaccinated mice (PBS) andmice vaccinated with the indicated dose of STARR™ SARS-CoV-2 RNA areshown.

FIG. 12 shows viral titers in lungs of unvaccinated mice (PBS) and micevaccinated with the indicated dose of STARR™ SARS-CoV-2 RNA followingchallenge with SARS-CoV-2.

FIG. 13 shows an RNA dose-dependent immunogenicity comparison betweenG614 and D614 SARS CoV-2 glycoprotein expressed from self-replicatingRNA.

FIG. 14 shows a schematic illustrating one aspect of STARR™ technologyand lipid-mediated delivery.

FIGS. 15A-15C show duration of luciferase reporter gene expression forself-replicating (replicon) RNA (STARR™), such as (15A) STARR™ FLuc,(15B) STARR™ FLuc IRES-E3L, and (15C) STARR™ FLuc IRES E3L (short 3′UTR) as compared to mRNA.

FIG. 16A-16D show results of Luminex Assay for anti-SARS-Cov-2 SpikeGlycoprotein IgG in two pre-clinical studies. BALB/c mice werevaccinated with increasing RNA doses of self-replicating RNA (SEQ IDNO:125) formulated as lyophilized lipid nanoparticles (LYO-LNP) andliquid (frozen) lipid nanoparticles (Liquid-LNP). (16A) First Study 0.2μg, (16B) First Study 2 μg, (16C) Second Study 0.2 μg, and (16D) SecondStudy 2 μg. Blood was collected and processed to scrum at various timespost-vaccination and evaluated for anti-SARS-CoV-2 spike glycoproteinIgG. Two way ANOVA, Tukey's multiple comparison post-test comparedLYO-LNP to Liquid-LNP where * p<0.0332, ** p<0.0021, *** p<0.0002, ****p<0.0001.

FIGS. 17A-17B show the Area Under the Curve (AUC) Analysis foranti-SARS-Cov-2 Spike Glycoprotein IgG (First and Second Study combineddata). IgG assay results were combined from two studies to evaluateself-replicating RNA (SEQ ID NO:125) formulated as lyophilized lipidnanoparticles (LYO-LNP) and liquid (frozen) lipid nanoparticles(Liquid-LNP) at (17A) 0.2 μg, and (17B) 2 μg. N=10/group. First StudyDay 19 and 31 results were combined with Second Study Day 20 and 30results, respectively, and an Area Under the Curve (AUC) analysis wasperformed. One way ANOVA, Sidak's multiple comparison post-test comparedLYO-LNP to Liquid-LNP and resulted in no statistical differences.

FIGS. 18A-18D shows characterization of STARR™ technology with fireflyluciferase transgene expression. (18A) Firefly luciferase (FLuc)expression from STARR™ Fluc, SINV FLuc, and mRNA FLuc was monitored upto day 28 by In Vivo Imaging System (IVIS). The average of total flux(p/s) from 6 injection sites in a mouse group was plotted at each timepoint with a standard error of mean, SEM. (18B) IVIS picture of threemice (6 injection sites) per group on day 14 is shown for each groupthat was administered with the test article labeled below the picture.(18C) Luciferase expression from mice that were intramuscularly injectedwith STARR™ FLuc was monitored by IVIS up to 63 days postadministration. (18D) Effect of prior administration of repliconbackbone was examined for STARR™ (upper panel) and SINV (lower panel).Replicon encoding FLuc was IM injected at 7 days post dose of repliconwith homologous backbone with an irrelevant gene/sequence (labeledSTARR™ irr or SINV irr) at day 0. As a reference, a mouse group with PBSadministration at day 0 was included in each of STARR™ and SINV group.

FIG. 19 shows that STARR™ elicits antigen-specific IFN-gamma response.Enzyme-linked immune absorbent spot ELISpot was used to count the numberof splenocytes that were specifically stimulated by an antigen peptideof the same amino acid sequence encoded in TA STARR™. Neither no peptide(cell only) nor irrelevant peptide (Bgal) did not elicit significantIFN-gamma from splenocytes from mice vaccinated with STARR™ FLuc or TASTARR™. Stimulation with AH1-A5 peptide resulted in the detection ofIFN-gamma-producing cells specifically from the mice that werevaccinated with TASTARR™. Concanavalin A (ConA) was used as a positivecontrol of IFN-gamma production.

FIGS. 20A-20F illustrate reduced tumor growth rate by TA STARR™vaccination in a CT26 syngeneic mouse model. CT26 murine colorectalcarcinoma cells (5×10⁵) were subcutaneously implanted in 10-week oldfemale BALB/c mice (n=8 per group). On days 1 and 8, the mice werevaccinated with STARR™ FLuc, a negative control, or TA STARR™, whichencodes AH1A5 epitope. Tumor growth was monitored in mice vaccinatedwith (20A) STARR™ FLuc without checkpoint inhibitor treatment; (20B)STARR™ FLuc with a combination anti-PD1/PDL1 treatment; (20C) STARR™FLuc with a combination anti-CTLA4 treatment; (20D) STARR™ vaccinewithout checkpoint inhibitor treatment; (20E) STARR™ vaccine with acombination treatment of anti-PD1 and anti-PDL1; and (20F) STARR™vaccine with a combination treatment of anti-CTLA4. The individual tumorgrowth curves from a mouse group that were administered with STARR™ FLucand TA STARR™ are shown in upper and lower panels, respectively.

FIG. 21 illustrates prolonged protection by combination treatment of TASTARR™ Vaccine with checkpoint inhibitors. Mice that were treated withTA STARR™ combined with anti-PD1/PDL1 or anti-CTLA4 were found to beresistant to tumor growth following the CT26 challenge at day 25 to 42.Naïve mice were used as a control for the CT26 tumor growth.

FIGS. 22A-22C show results from AH1-tetramer staining of CD8+ T-cells inthe form of (22A) a graph and (22B and 22C) plots. Splenocytes from themice group with combination treatment of TA STARR™ and anti-PD1/PDL1 atday 42 were stained with AH1 (H-2Ld)-tetramer. The staining was specificto CD8+ T cells from the mouse group with TA STARR™ treatment, and thepopulation represented 9-17% of total CD8+ T cells from the splenocytes.

FIG. 23 shows HA titers obtained for self-replicating RNA (STARR™) andmRNA constructs encoding the hemagglutinin of influenza virusA/California/07/2009 (H1N1).

FIGS. 24A-24B show RNA replication levels (FIG. 24A) and luciferasereporter gene expression levels (FIG. 24B) for the indicatedself-replicating (replicon) RNAs as compared to mRNA.

DETAILED DESCRIPTION

The present disclosure relates to self-replicating RNAs and nucleicacids encoding the same for expression of transgenes such as antigenicproteins and tumor antigens, for example. Also provided herein aremethods of administration (e.g., to a host, such as a mammalian subject)of self-replicating RNAs, whereby the self-replicating RNA is translatedin vivo and the heterologous protein-coding sequence is expressed and,e.g., can elicit an immune response to the heterologous protein-codingsequence in the recipient or provide a therapeutic effect, where theheterologous protein-coding sequence is a therapeutic protein.Self-replicating RNAs provided herein are useful as vaccines that can berapidly generated and that can be effective at low and/or single doses.The present disclosure further relates to methods of inducing an immuneresponse using self-replicating RNAs provided herein.

In some embodiments, an immune response can be elicited againstCoronavirus: immunogens that include, but are not limited to, thosederived from a SARS coronavirus, avian infectious bronchitis (IBV),Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritisvirus (TGEV). The coronavirus immunogen may be a spike polypeptide.

Self-replicating RNAs are described, for example, in U.S. 2018/0036398,the contents of which are incorporated by reference in their entirety.

Definitions

As used herein, the term “fragment,” when referring to a protein ornucleic acid, for example, means any shorter sequence than thefull-length protein or nucleic acid. Accordingly, any sequence of anucleic acid or protein other than the full-length nucleic acid orprotein sequence can be a fragment. In some aspects, a protein fragmentincludes an epitope. In other aspects, a protein fragment is an epitope.

As used herein, the term “nucleic acid” refers to any deoxyribonucleicacid (DNA) molecule, ribonucleic acid (RNA) molecule, or nucleic acidanalogues. A DNA or RNA molecule can be double-stranded orsingle-stranded and can be of any size. Exemplary nucleic acids include,but are not limited to, chromosomal DNA, plasmid DNA, cDNA, cell-freeDNA (cfDNA), mitochondrial DNA, chloroplast DNA, viral DNA, mRNA, tRNA,rRNA, long non-coding RNA, siRNA, micro RNA (miRNA or miR), hnRNA, andviral RNA. Exemplary nucleic analogues include peptide nucleic acid,morpholino- and locked nucleic acid, glycol nucleic acid, and threosenucleic acid. As used herein, the term “nucleic acid molecule” is meantto include fragments of nucleic acid molecules as well as anyfull-length or non-fragmented nucleic acid molecule, for example. Asused herein, the terms “nucleic acid” and “nucleic acid molecule” can beused interchangeably, unless context clearly indicates otherwise.

As used herein, the term “protein” refers to any polymeric chain ofamino acids. The terms “peptide” and “polypeptide” can be usedinterchangeably with the term protein, unless context clearly indicatesotherwise, and can also refer to a polymeric chain of amino acids. Theterm “protein” encompasses native or artificial proteins, proteinfragments and polypeptide analogs of a protein sequence. A protein maybe monomeric or polymeric. The term “protein” encompasses fragments andvariants (including fragments of variants) thereof, unless otherwisecontradicted by context.

In general, “sequence identity” or “sequence homology,” which can beused interchangeably, refer to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Typically, techniques fordetermining sequence identity include determining the nucleotidesequence of a polynucleotide and/or determining the amino acid sequenceencoded thereby or the amino acid sequence of a polypeptide, andcomparing these sequences to a second nucleotide or amino acid sequence.As used herein, the term “percent (%) sequence identity” or “percent (%)identity,” also including “homology,” refers to the percentage of aminoacid residues or nucleotides in a sequence that are identical with theamino acid residues or nucleotides in a reference sequence afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Thus, twoor more sequences (polynucleotide or amino acid) can be compared bydetermining their “percent identity,” also referred to as “percenthomology.” The percent identity to a reference sequence (e.g., nucleicacid or amino acid sequences), which may be a sequence within a longermolecule (e.g., polynucleotide or polypeptide), may be calculated as thenumber of exact matches between two optimally aligned sequences dividedby the length of the reference sequence and multiplied by 100. Percentidentity may also be determined, for example, by comparing sequenceinformation using the advanced BLAST computer program, including version2.2.9, available from the National Institutes of Health. The BLASTprogram is based on the alignment method of Karlin and Altschul, Proc.Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul etal., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl.Acad. sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997). Briefly, the BLAST program defines identity asthe number of identical aligned symbols (i.e., nucleotides or aminoacids), divided by the total number of symbols in the shorter of the twosequences. The program may be used to determine percent identity overthe entire length of the sequences being compared. Default parametersare provided to optimize searches with short query sequences, forexample, with the blastp program. The program also allows use of an SEGfilter to mask-off segments of the query sequences as determined by theSEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163(1993). Ranges of desired degrees of sequence identity are approximately80% to 100% and integer values in between. Percent identities between areference sequence and a claimed sequence can be at least 80%, at least85%, at least 90%, at least 95%, at least 98%, at least 99%, at least99.5%, or at least 99.9%. In general, an exact match indicates 100%identity over the length of the reference sequence. Additional programsand methods for comparing sequences and/or assessing sequence identityinclude the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needlealigner available at cbi.ac.uk/Tools/psa/emboss needle/, optionally withdefault settings), the Smith-Waterman algorithm (see, e.g., the EMBOSSWater aligner available at ebi.ac.uk/Tools/psa/emboss water/, optionallywith default settings), the similarity search method of Pearson andLipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or computer programswhich use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N andTFASTA in Wisconsin Genetics Software Package, Genetics Computer Group.575 Science Drive, Madison, Wis.). In some aspects, reference to percentsequence identity refers to sequence identity as measured using BLAST(Basic Local Alignment Search Tool). In other aspects, ClustalW is usedfor multiple sequence alignment. Optimal alignment may be assessed usingany suitable parameters of a chosen algorithm, including defaultparameters.

As used herein, the term “drug” or “medicament,” means a pharmaceuticalformulation or composition as described herein.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. Thus, forexample, references to “the method” includes one or more methods, and/orsteps of the type described herein which will become apparent to thosepersons skilled in the art upon reading this disclosure and so forth.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of +20%, or ±10%, or ±5%, or even ±1% from the specifiedvalue, as such variations are appropriate for the disclosed methods orto perform the disclosed methods.

The term “expression” refers to the process by which a nucleic acidsequence or a polynucleotide is transcribed from a DNA template (such asinto mRNA or other RNA transcript) and/or the process by which atranscribed mRNA or other RNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.”

As used herein, the terms “self-replicating RNA,” “self-transcribing andself-replicating RNA,” “self-amplifying RNA (saRNA),” and “replicon” maybe used interchangeably, unless context clearly indicates otherwise.Generally, the term “replicon” or “viral replicon” refers to aself-replicating subgenomic RNA derived from a viral genome thatincludes viral genes encoding non-structural proteins important forviral replication and that lacks viral genes encoding structuralproteins. A self-replicating RNA can encode further subgenomic RNAs thatare not able to self-replicate.

As used herein, “operably linked,” “operable linkage,” “operativelylinked,” or grammatical equivalents thereof refer to juxtaposition ofgenetic elements, e.g., a promoter, an enhancer, a polyadenylationsequence, etc., wherein the elements are in a relationship permittingthem to operate in the expected manner. For instance, a regulatoryelement, which can comprise promoter and/or enhancer sequences, isoperatively linked to a coding region if the regulatory element helpsinitiate transcription of the coding sequence. There may be interveningresidues between the regulatory element and coding region so long asthis functional relationship is maintained.

Nucleic Acid Molecules

In some embodiments, provided herein are nucleic acid moleculescomprising: (i) a first polynucleotide encoding one or more viralreplication proteins, wherein the first polynucleotide iscodon-optimized as compared to a wild-type polynucleotide encoding theone or more viral replication proteins; and (ii) a second polynucleotidecomprising a first transgene encoding a first antigenic protein or afragment thereof, wherein the first antigenic protein is a coronavirusprotein.

An RNA molecule can encode a single polypeptide immunogen or multiplepolypeptides. Multiple immunogens can be presented as a singlepolypeptide immunogen (fusion polypeptide) or as separate polypeptides.If immunogens are expressed as separate polypeptides from a repliconthen one or more of these may be provided with an upstream IRES or anadditional viral promoter element. Alternatively, multiple immunogensmay be expressed from a polyprotein that encodes individual immunogensfused to a short autocatalytic protease (e.g. foot-and-mouth diseasevirus 2A protein), or as inteins.

Also provided herein, in some embodiments, are nucleic acid moleculescomprising: (i) a first polynucleotide comprising a sequence having atleast 80% identity to a sequence of SEQ ID NO:72; and (ii) a secondpolynucleotide comprising a first transgene encoding a first antigenicprotein or a fragment thereof.

Codon Optimization

In some embodiments, first polynucleotides of nucleic acid moleculesprovided herein encoding one or more viral replication proteins includecodon-optimized sequences. As used herein, the term “codon-optimized”means a polynucleotide, nucleic acid sequence, or coding sequence hasbeen redesigned as compared to a wild-type or reference polynucleotide,nucleic acid sequence, or coding sequence by choosing different codonswithout altering the amino acid sequence of the encoded protein.Accordingly, codon-optimization generally refers to replacement ofcodons with synonymous codons to optimize expression of a protein whilekeeping the amino acid sequence of the translated protein the same.Codon optimization of a sequence can increase protein expression levels(Gustafsson et al., Codon bias and heterologous protein expression.2004, Trends Biotechnol 22: 346-53) of the encoded proteins, forexample, and provide other advantages. Variables such as codon usagepreference as measured by codon adaptation index (CAI), for example, thepresence or frequency of U and other nucleotides, mRNA secondarystructures, cis-regulatory sequences, GC content, and other variablesmay correlate with protein expression levels (Villalobos et al., GeneDesigner: a synthetic biology tool for constructing artificial DNAsegments. 2006, BMC Bioinformatics 7:285).

Any method of codon optimization can be used to codon optimizepolynucleotides and nucleic acid molecules provided herein, and anyvariable can be altered by codon optimization. Accordingly, anycombination of codon optimization methods can be used. Exemplary methodsinclude the high codon adaptation index (CAI) method, the Low U method,and others. The CAI method chooses a most frequently used synonymouscodon for an entire protein coding sequence. As an example, the mostfrequently used codon for each amino acid can be deduced from 74,218protein-coding genes from a human genome. The Low U method targetsU-containing codons that can be replaced with a synonymous codon withfewer U moieties, generally without changing other codons. If there ismore than one choice for replacement, the more frequently used codon canbe selected. Any polynucleotide, nucleic acid sequence, or codonsequence provided herein can be codon-optimized.

In some embodiments, the nucleotide sequence of any region of the RNA orDNA templates described herein may be codon optimized. Preferably, theprimary cDNA template may include reducing the occurrence or frequencyof appearance of certain nucleotides in the template strand. Forexample, the occurrence of a nucleotide in a template may be reduced toa level below 25% of said nucleotides in the template. In furtherexamples, the occurrence of a nucleotide in a template may be reduced toa level below 20% of said nucleotides in the template. In some examples,the occurrence of a nucleotide in a template may be reduced to a levelbelow 16% of said nucleotides in the template. Preferably, theoccurrence of a nucleotide in a template may be reduced to a level below15%, and preferably may be reduced to a level below 12% of saidnucleotides in the template.

In some embodiments, the nucleotide reduced is uridine. For example, thepresent disclosure provides nucleic acids with altered uracil contentwherein at least one codon in the wild-type sequence has been replacedwith an alternative codon to generate a uracil-altered sequence. Altereduracil sequences can have at least one of the following properties:

-   -   (i) an increase or decrease in global uracil content (i.e., the        percentage of uracil of the total nucleotide content in the        nucleic acid of a section of the nucleic acid, e.g., the open        reading frame);    -   (ii) an increase or decrease in local uracil content (i.e.,        changes in uracil content are limited to specific subsequences);    -   (iii) a change in uracil distribution without a change in the        global uracil content;    -   (iv) a change in uracil clustering (e.g., number of clusters,        location of clusters, or distance between clusters); or    -   (v) combinations thereof.

In some embodiments, the percentage of uracil nucleobases in the nucleicacid sequence is reduced with respect to the percentage of uracilnucleobases in the wild-type nucleic acid sequence. For example, 30% ofnucleobases may be uracil in the wild-type sequence but the nucleobasesthat are uracil are preferably lower than 15%, preferably lower than 12%and preferably lower than 10% of the nucleobases in the nucleic acidsequences of the disclosure. The percentage uracil content can bedetermined by dividing the number of uracil in a sequence by the totalnumber of nucleotides and multiplying by 100.

In some embodiments, the percentage of uracil nucleobases in asubsequence of the nucleic acid sequence is reduced with respect to thepercentage of uracil nucleobases in the corresponding subsequence of thewild-type sequence. For example, the wild-type sequence may have a5′-end region (e.g., 30 codons) with a local uracil content of 30%, andthe uracil content in that same region could be reduced to preferably15% or lower, preferably 12% or lower and preferably 10% or lower in thenucleic acid sequences of the disclosure. These subsequences can also bepart of the wild-type sequences of the heterologous 5′ and 3′ UTRsequences of the present disclosure.

In some embodiments, codons in the nucleic acid sequence of thedisclosure reduce or modify, for example, the number, size, location, ordistribution of uracil clusters that could have deleterious effects onprotein translation. Although lower uracil content is desirable incertain aspects, the uracil content, and in particular the local uracilcontent, of some subsequences of the wild-type sequence can be greaterthan the wild-type sequence and still maintain beneficial features(e.g., increased expression).

In some embodiments, the uracil-modified sequence induces a lowerToll-Like Receptor (TLR) response when compared to the wild-typesequence. Several TLRs recognize and respond to nucleic acids.Double-stranded (ds)RNA, a frequent viral constituent, has been shown toactivate TLR3. Single-stranded (ss)RNA activates TLR7. RNAoligonucleotides, for example RNA with phosphorothioate internucleotidelinkages, are ligands of human TLR8. DNA containing unmethylated CpGmotifs, characteristic of bacterial and viral DNA, activate TLR9.

As used herein, the term “TLR response” is defined as the recognition ofsingle-stranded RNA by a TLR7 receptor, and preferably encompasses thedegradation of the RNA and/or physiological responses caused by therecognition of the single-stranded RNA by the receptor. Methods todetermine and quantify the binding of an RNA to a TLR7 are known in theart. Similarly, methods to determine whether an RNA has triggered aTLR7-mediated physiological response (e.g., cytokine secretion) are wellknown in the art. In some embodiments, a TLR response can be mediated byTLR3, TLR8, or TLR9 instead of TLR7. Suppression of TLR7-mediatedresponse can be accomplished via nucleoside modification. RNA undergoesover a hundred different nucleoside modifications in nature. Human rRNA,for example, has ten times more pseudouracil (‘P) and 25 times more2′-O-methylated nucleosides than bacterial rRNA. Bacterial RNA containsno nucleoside modifications, whereas mammalian RNAs have modifiednucleosides such as 5-methylcytidine (m5C), N6-methyladenosine (m6A),inosine and many 2′-O-methylated nucleosides in addition toN7-methylguanosine (m7G).

In some embodiments, the uracil content of polynucleotides disclosedherein is less than about 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%,41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%,27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the totalnucleobases in the sequence in the reference sequence. In someembodiments, the uracil content of polynucleotides disclosed herein isbetween about 5% and about 25%. In some embodiments, the uracil contentof polynucleotides disclosed herein is between about 15% and about 25%.

In some embodiments, first polynucleotides of nucleic acid moleculesprovided herein comprise a sequence having at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, atleast 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least99.7%, at least 99.8%, at least 99.9%, and any number or range inbetween, identity to a sequence of SEQ ID NO:72. In some embodiments,first polynucleotides of nucleic acid molecules provided herein comprisea sequence of SEQ ID NO:72.

In some aspects, first polynucleotides and second polynucleotides ofnucleic acid molecules provided herein are included in the same (i.e., asingle) or in separate nucleic acid molecules. Generally, firstpolynucleotides and second polynucleotides of nucleic acid moleculesprovided herein are included in a single nucleic acid molecule. In oneaspect, the first polynucleotide is located 5′ of the secondpolynucleotide. In one aspect, first polynucleotides and secondpolynucleotides of nucleic acid molecules provided herein are includedin separate nucleic acid molecules. In yet another aspect, firstpolynucleotides and second polynucleotides are included in two separatenucleic acid molecules.

In some aspects, first polynucleotides and second polynucleotides areincluded in the same (i.e., a single) nucleic acid molecule. Firstpolynucleotides and second polynucleotides of nucleic acid moleculesprovided herein can be contiguous, i.e., adjacent to each other withoutnucleotides in between. In one aspect, an intergenic region is locatedbetween the first polynucleotide and the second polynucleotide. Inanother aspect, the intergenic region located between the firstpolynucleotide and the second polynucleotide is a second intergenicregion, with a first intergenic region included in the firstpolynucleotide as described below. As used herein, the terms “intergenicregion” and intergenic sequence” can be used interchangeably, unlesscontext clearly indicates otherwise.

An intergenic region located between the first polynucleotide and thesecond polynucleotide can be of any length and can have any nucleotidesequence. As an example, the intergenic region between the firstpolynucleotide and the second polynucleotide can include about onenucleotide, about two nucleotides, about three nucleotides, about fournucleotides, about five nucleotides, about six nucleotides, about sevennucleotides, about eight nucleotides, about nine nucleotides, about tennucleotides, about 11 nucleotides, about 12 nucleotides, about 13nucleotides, about 14 nucleotides, about 15 nucleotides, about 16nucleotides, about 17 nucleotides, about 18 nucleotides, about 19nucleotides, about 20 nucleotides, about 21 nucleotides, about 22nucleotides, about 23 nucleotides, about 24 nucleotides, about 25nucleotides, about 26 nucleotides, about 27 nucleotides, about 28nucleotides, about 29 nucleotides, about 30 nucleotides, about 31nucleotides, about 32 nucleotides, about 33 nucleotides, about 34nucleotides, about 35 nucleotides, about 36 nucleotides, about 37nucleotides, about 38 nucleotides, about 39 nucleotides, about 40nucleotides, about 41 nucleotides, about 42 nucleotides, about 43nucleotides, about 44 nucleotides, about 45 nucleotides, about 46nucleotides, about 47 nucleotides, about 48 nucleotides, about 49nucleotides, about 50 nucleotides, about 60 nucleotides, about 70nucleotides, about 80 nucleotides, about 90 nucleotides, about 100nucleotides, about 125 nucleotides, about 150 nucleotides, about 175nucleotides, about 200 nucleotides, about 250 nucleotides, about 300nucleotides, about 350 nucleotides, about 400 nucleotides, about 450nucleotides, about 500 nucleotides, about 600 nucleotides, about 700nucleotides, about 800 nucleotides, about 1,000 nucleotides, about 1,500nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides,about 4,500 nucleotides, about 5,000 nucleotides, about 6,000nucleotides, about 7,000 nucleotides, about 8,000 nucleotides, about9,000 nucleotides, about 10,000 nucleotides, and any number or range inbetween. In one aspect, the intergenic region between first and secondpolynucleotides includes about 10-100 nucleotides, about 10-200nucleotides, about 10-300 nucleotides, about 10-400 nucleotides, orabout 10-500 nucleotides. In another aspect, the intergenic regionbetween first and second polynucleotides includes about 1-10nucleotides, about 1-20 nucleotides, about 1-30 nucleotides, about 1-40nucleotides, or about 1-50 nucleotides. In yet another aspect, theregion includes about 44 nucleotides. In one aspect, the intergenicregion between first and second polynucleotides of nucleic acidmolecules provided herein is a second intergenic region.

In one aspect, the intergenic region between first and secondpolynucleotides includes a viral sequence. The intergenic region betweenfirst and second polynucleotides can include a sequence from any virus,such as alphaviruses and rubiviruses, for example. In one aspect, theintergenic region between the first polynucleotide and the secondpolynucleotide comprises an alphavirus sequence, such as a sequence fromVenezuelan Equine Encephalitis Virus (VEEV), Eastern Equine EncephalitisVirus (EEEV), Everglades Virus (EVEV), Mucambo Virus (MUCV), SemlikiForest Virus (SFV), Pixuna Virus (PIXV), Middleburg Virus (MIDV),Chikungunya Virus (CHIKV), O'Nyong-Nyong Virus (ONNV), Ross River Virus(RRV), Barmah Forest Virus (BFV), Getah Virus (GETV), Sagiyama Virus(SAGV), Bebaru Virus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV),Sindbis Virus (SINV), Aura Virus (AURAV), Whataroa Virus (WHAV), BabankiVirus (BABV), Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus(WEEV), Highland J Virus (HJV), Fort Morgan Virus (FMV), Ndumu Virus(NDUV), Salmonid Alphavirus (SAV), Buggy Creek Virus (BCRV), or anycombination thereof. In another aspect, the intergenic region betweenfirst and second polynucleotides comprises a sequence from VenezuelanEquine Encephalitis Virus (VEEV). In yet another aspect, the intergenicregion between first and second polynucleotides comprises a sequencehaving at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, atleast 99.6%, at least 99.7%, at least 99.8%, at least 99.9% h, and anynumber or range in between, identity to SEQ ID NO:77. In a furtheraspect, the intergenic region between first and second polynucleotidescomprises a sequence of SEQ ID NO:77. In yet a further aspect, theintergenic region between first and second polynucleotides is a secondintergenic region comprising a sequence having at least 85% identity toSEQ ID NO:77.

Natural and Modified Nucleotides

A self-replicating RNA of the disclosure can comprise one or morechemically modified nucleotides. Examples of nucleic acid monomersinclude non-natural, modified, and chemically-modified nucleotides,including any such nucleotides known in the art. Nucleotides can beartificially modified at either the base portion or the sugar portion.In nature, most polynucleotides comprise nucleotides that are“unmodified” or “natural” nucleotides, which include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). These bases are typically fixed to a riboseor deoxy ribose at the 1′ position. The use of RNA polynucleotidescomprising chemically modified nucleotides have been shown to improveRNA expression, expression rates, half-life and/or expressed proteinconcentrations. RNA polynucleotides comprising chemically modifiednucleotides have also been useful in optimizing protein localizationthereby avoiding deleterious bio-responses such as immune responsesand/or degradation pathways.

Examples of modified or chemically-modified nucleotides include5-hydroxycytidines, 5-alkylcytidines, 5-hydroxyalkylcytidines,5-carboxycytidines, 5-formylcytidines, 5-alkoxycytidines,5-alkynylcytidines, 5-halocytidines, 2-thiocytidines, N4-alkylcytidines,N4-aminocytidines, N4-acetylcytidines, and N4,N4-dialkylcytidines.

Examples of modified or chemically-modified nucleotides include5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine,5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine,5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine;N4-methylcytidine, N4-aminocytidine, N4-acetylcytidine, andN4,N4-dimethylcytidine.

Examples of modified or chemically-modified nucleotides include5-hydroxyuridines, 5-alkyluridines, 5-hydroxyalkyluridines,5-carboxyuridines, 5-carboxyalkylesteruridines, 5-formyluridines,5-alkoxyuridines, 5-alkynyluridines, 5-halouridines, 2-thiouridines, and6-alkyluridines.

Examples of modified or chemically-modified nucleotides include5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine,5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine,5-methoxyuridine (also referred to herein as “5MeOU”),5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine,2-thiouridine, and 6-methyluridine.

Examples of modified or chemically-modified nucleotides include5-mcthoxycarbonylmethyl-2-thiouridine,5-methylaminomethyl-2-thiouridine, 5-carbamoylmethyluridine,5-carbamoylmethyl-2′-O-methyluridine,1-methyl-3-(3-amino-3-carboxypropy)pseudouridine,5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine,5-methyldihydrouridine, 5-taurinomethyluridine,5-taurinomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine,2′-O-methylpseudouridine, 2-thio-2′O-methyluridine, and3,2′-O-dimethyluridine.

Examples of modified or chemically-modified nucleotides includeN6-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine,7-deazaadenosine, 8-oxoadenosine, 8-bromoadenosine,2-methylthio-N6-methyladenosine, N6-isopentenyladenosine,2-methylthio-N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyl-adenosine,N6-methyl-N6-threonylcarbamoyl-adenosine,2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyladenosine,N6-hydroxynorvalylcarbamoyladenosine,2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine,7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine,alpha-thio-adenosine, 2′-O-methyl-adenosine, N6,2′-O-dimethyl-adenosine,N6,N6,2′-O-trimethyl-adenosine, 1,2′-O-dimethyl-adenosine,2′-O-ribosyladenosine, 2-amino-N6-methyl-purine, 1-thio-adenosine,2′-F-ara-adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, andN6-(19-amino-pentaoxanonadecyl)-adenosine.

Examples of modified or chemically-modified nucleotides includeN1-alkylguanosincs, N2-alkylguanosines, thienoguanosines,7-deazaguanosines, 8-oxoguanosines, 8-bromoguanosines,06-alkylguanosines, xanthosines, inosines, and N1-alkylinosines.

Examples of modified or chemically-modified nucleotides includeN1-methylguanosine, N2-methylguanosine, thienoguanosine,7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, 06-methylguanosine,xanthosine, inosine, and N1-methylinosine.

Examples of modified or chemically-modified nucleotides includepseudouridines. Examples of pseudouridines includeN1-alkylpseudouridines, N1-cycloalkylpseudouridines,N1-hydroxypseudouridines, N1-hydroxyalkylpseudouridines,N1-phenylpseudouridines, N1-phenylalkylpseudouridines,N1-aminoalkylpseudouridines, N3-alkylpseudouridines,N6-alkylpseudouridines, N6-alkoxypseudouridines,N6-hydroxypseudouridines, N6-hydroxyalkylpseudouridines,N6-morpholinopseudouridines, N6-phenylpseudouridines, andN6-halopseudouridines. Examples of pseudouridines includeN1-alkyl-N6-alkylpseudouridines, N1-alkyl-N6-alkoxypseudouridines,N1-alkyl-N6-hydroxypseudouridines,N1-alkyl-N6-hydroxyalkylpseudouridines,N1-alkyl-N6-morpholinopseudouridines, N1-alkyl-N6-phenylpseudouridines,and N1-alkyl-N6-halopseudouridines. In these examples, the alkyl,cycloalkyl, and phenyl substituents may be unsubstituted, or furthersubstituted with alkyl, halo, haloalkyl, amino, or nitro substituents.

Examples of pseudouridines include N1-methylpseudouridine (also referredto herein as “NIMPU”), N1-ethylpseudouridine, N1-propylpseudouridine,N1-cyclopropylpseudouridine, N1-phenylpseudouridine,N1-aminomethylpseudouridine, N3-methylpseudouridine,N1-hydroxypseudouridine, and N1-hydroxymethylpseudouridine.

Examples of nucleic acid monomers include modified andchemically-modified nucleotides, including any such nucleotides known inthe art.

Examples of modified and chemically-modified nucleotide monomers includeany such nucleotides known in the art, for example, 2′-O-methylribonucleotides, 2′-O-methyl purine nucleotides, 2′-deoxy-2′-fluororibonucleotides, 2′-deoxy-2′-fluoro pyrimidine nucleotides, 2′-deoxyribonucleotides, 2′-deoxy purine nucleotides, universal basenucleotides, 5-C-methyl-nucleotides, and inverted deoxyabasic monomerresidues.

Examples of modified and chemically-modified nucleotide monomers include3′-end stabilized nucleotides, 3′-glyceryl nucleotides, 3′-invertedabasic nucleotides, and 3′-inverted thymidine.

Examples of modified and chemically-modified nucleotide monomers includelocked nucleic acid nucleotides (LNA),2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides, 2′-methoxyethoxy(MOE) nucleotides, 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides,and 2′-O-methyl nucleotides. In an exemplary embodiment, the modifiedmonomer is a locked nucleic acid nucleotide (LNA).

Examples of modified and chemically-modified nucleotide monomers include2′,4′-constrained 2′-O-methoxyethyl (cMOE) and 2′-O-Ethyl (cEt) modifiedDNAs.

Examples of modified and chemically-modified nucleotide monomers include2′-amino nucleotides, 2′-O-amino nucleotides, 2′-C-allyl nucleotides,and 2′-O-allyl nucleotides.

Examples of modified and chemically-modified nucleotide monomers includeN6-methyladenosine nucleotides.

Examples of modified and chemically-modified nucleotide monomers includenucleotide monomers with modified bases 5-(3-amino)propyluridine,5-(2-mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or7-deazaadenosine.

Examples of modified and chemically-modified nucleotide monomers include2′-O-aminopropyl substituted nucleotides.

Examples of modified and chemically-modified nucleotide monomers includereplacing the 2′-OH group of a nucleotide with a 2′-R, a 2′-OR, a2′-halogen, a 2′-SR, or a 2′-amino, where R can be H, alkyl, alkenyl, oralkynyl.

Example of base modifications described above can be combined withadditional modifications of nucleoside or nucleotide structure,including sugar modifications and linkage modifications. Certainmodified or chemically-modified nucleotide monomers may be found innature.

Preferred nucleotide modifications include N1-methylpseudouridine and5-methoxyuridine.

Viral Replication Proteins and Polynucleotides Encoding them

Provided herein, in some embodiments, are nucleic acid moleculescomprising a first polynucleotide encoding one or more viral replicationproteins. As used herein, the term “replication protein” or “viralreplication protein” refers to any protein or any protein subunit of aprotein complex that functions in replication of a viral genome.Generally, viral replication proteins are non-structural proteins. Viralreplication proteins encoded by nucleic acid molecules provided hereincan function in the replication of any viral genome. The viral genomecan be a single-stranded positive-sense RNA genome, a single-strandednegative-sense RNA genome, a double-stranded RNA genome, asingle-stranded positive-sense DNA genome, a single-strandednegative-sense DNA genome, or a double-stranded DNA genome. Viralgenomes can include a single nucleic acid molecule or more than onenucleic acid molecule. Nucleic acid molecules provided herein can encodeone or more viral replication proteins from any virus or virus family,including animal viruses and plant viruses, for example. Viralreplication proteins encoded by first polynucleotides included innucleic acid molecules provided herein can be expressed fromself-replicating RNA.

First polynucleotide sequences of nucleic acid molecules provided hereincan encode one or more togavirus replication proteins. In some aspects,the one or more viral replication proteins encoded by firstpolynucleotides of nucleic acid molecules provided herein are alphavirusproteins. In some embodiments, the one or more viral replicationproteins encoded by first polynucleotides of nucleic acid moleculesprovided herein are rubivirus proteins. First polynucleotide sequencesof nucleic acid molecules provided herein can encode any alphavirusreplication protein and any rubivirus replication protein. Exemplaryreplication proteins from alphaviruses include proteins from VenezuelanEquine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus(EEEV), Everglades Virus (EVEV), Mucambo Virus (MUCV), Semliki ForestVirus (SFV), Pixuna Virus (PIXV), Middleburg Virus (MIDV), ChikungunyaVirus (CHIKV), O'Nyong-Nyong Virus (ONNV), Ross River Virus (RRV),Barmah Forest Virus (BFV), Getah Virus (GETV), Sagiyama Virus (SAGV),Bebaru Virus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), SindbisVirus (SINV), Aura Virus (AURAV), Whataroa Virus (WHAV), Babanki Virus(BABV), Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus(WEEV), Highland J Virus (HJV), Fort Morgan Virus (FMV), Ndumu Virus(NDUV), Salmonid Alphavirus (SAV), Buggy Creek Virus (BCRV), and anycombination thereof. Exemplary rubivirus replication proteins includeproteins from rubella virus.

Viral replication proteins encoded by first polynucleotides of nucleicacid molecules provided herein can be expressed as one or morepolyproteins or as separate or single proteins. Generally, polyproteinsare precursor proteins that are cleaved to generate individual orseparate proteins. Accordingly, proteins derived from a precursorpolyprotein can be expressed from a single open reading frame (ORF). Asused herein, the term “ORF” refers to a nucleotide sequence that beginswith a start codon, generally ATG, and that ends with a stop codon, suchas TAA, TAG, or TGA, for example. It will be appreciated that T ispresent in DNA, while U is present in RNA. Accordingly, a start codon ofATG in DNA corresponds to AUG in RNA, and the stop codons TAA, TAG, andTGA in DNA correspond to UAA, UAG, and UGA in RNA. It will further beappreciated that for any sequence provided in the present disclosure, Tis present in DNA, while U is present in RNA. Accordingly, for anysequence provided herein, T present in DNA is substituted with U for anRNA molecule, and U present in RNA is substituted with T for a DNAmolecule.

The protease cleaving a polyprotein can be a viral protease or acellular protease. In some aspects, the first polynucleotide of nucleicacid molecules provided herein encodes a polyprotein comprising analphavirus nsP1 protein, an alphavirus nsP2 protein, an alphavirus nsP3protein, an alphavirus nsP4 protein, or any combination thereof. Inother aspects, the first polynucleotide of nucleic acid moleculesprovided herein encodes a polyprotein comprising an alphavirus nsP1protein, an alphavirus nsP2 protein, an alphavirus nsP3 protein, or anycombination thereof, and an alphavirus nsP4 protein. In some aspects,the polyprotein is a VEEV polyprotein. In other aspects, the alphavirusnsP1, nsP2, nsP3, and nsP4 proteins are VEEV proteins.

In one aspect, first polynucleotides of nucleic acid molecules providedherein lack a stop codon between sequences encoding an nsP3 protein andan nsP4 protein. Accordingly, in some aspects, first polynucleotides ofnucleic acid molecules provided herein encode a P1234 polyproteincomprising nsP1, nsP2, nsP3, and nsP4. First polynucleotides of nucleicacid molecules provided herein can also include a stop codon betweensequences encoding an nsP3 and an nsP4 protein. Accordingly, in someaspects, first polynucleotides of nucleic acid molecules provided hereinencode a P123 polyprotein comprising nsP1, nsP2, and nsP3 and a P1234polyprotein comprising nsP1, nsP2, nsP3, and nsP4 as a result of stopcodon readthrough, for example. In other aspects, first polynucleotidesof nucleic acid molecules provided herein encode a polyprotein having atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and anynumber or range in between, identity to a sequence of SEQ ID NO:79. Insome embodiments, first polynucleotides of nucleic acid moleculesprovided herein encode a polyprotein having a sequence of SEQ ID NO:79.Further exemplary polyproteins comprise a sequence of SEQ ID NO:80 orSEQ ID NO:81. In one aspect, nsP2 and nsP3 proteins include mutations.Exemplary mutations include G1309R and S1583G mutations of VEEVproteins. In another aspect, the nsP1, nsP2, and nsP4 proteins are VEEVproteins, and the nsP3 protein is a chikungunya virus (CHIKV) nsP3protein.

In some aspects, first polynucleotides of nucleic acid moleculesprovided herein can include a first intergenic region. In some aspects,the first intergenic region is located between a sequence encoding apolyprotein comprising an alphavirus nsP1 protein, an alphavirus nsP2protein, an alphavirus nsP3 protein, or any combination thereof, and asequence encoding an alphavirus nsP4 protein. A first intergenic regioncan comprise any sequence, such as any viral or non-viral sequence. Inone aspect, the first intergenic region comprises a viral sequence. Inanother aspect, the first intergenic region comprises an alphavirussequence. In yet another aspect, the alphavirus is VEEV. In one aspect,nsP2 and nsP3 proteins include mutations. Exemplary mutations includeG1309R and S1583G mutations of VEEV proteins. In another aspect, thensP1, nsP2, and nsP4 proteins are VEEV proteins, and the nsP3 protein isa chikungunya virus (CHIKV) nsP3 protein.

In some embodiments, the first polynucleotide may comprise a sequencehaving at least 80% identity to a sequence of SEQ ID NO:72.

In some embodiments, the nucleic acid molecule described herein mayfurther comprise a second polynucleotide comprising a first transgeneencoding a first antigenic protein or fragment thereof, wherein thefirst antigenic protein is a coronavirus protein. In specificembodiments, the antigenic protein may be a SARS-CoV-2 protein. Inspecific embodiments, the antigenic protein is a SARS-CoV-2 spikeglycoprotein. In specific embodiments, the SARS-CoV-2 spike glycoproteinis a wild-type SARS-CoV-2 spike glycoprotein having an amino acidsequence of SEQ ID NO:123.

In some embodiments, the second polynucleotide comprises a sequencehaving at least 85% identity to a sequence of SEQ ID NO:121 or SEQ IDNO:122.

5′ Untranslated Region (5′ UTR)

Nucleic acid molecules provided herein can further comprise untranslatedregions (UTRs). Untranslated regions, including 5′ UTRs and 3′ UTRs, forexample, can affect RNA stability and/or efficiency of RNA translation,such as translation of cellular and viral mRNAs, for example. 5′ UTRsand 3′ UTRs can also affect stability and translation of viral genomicRNAs and self-replicating RNAs, including virally derivedself-replicating RNAs or replicons. Exemplary viral genomic RNAs whosestability and/or efficiency of translation can be affected by 5′ UTRsand 3′ UTRs include the genome nucleic acid of positive-sense RNAviruses. Both genome nucleic acid of positive-sense RNA viruses andself-replicating RNAs, including virally derived self-replicating RNAsor replicons, can be translated upon infection or introduction into acell.

In some aspects, nucleic acid molecules provided herein further includea 5′ untranslated region (5′ UTR). Any 5′ UTR sequence can be includedin nucleic acid molecules provided herein. In some embodiments, nucleicacid molecules provided herein include a viral 5′ UTR. In one aspect,nucleic acid molecules provided herein include a non-viral 5′ UTR. Anynon-viral 5′ UTR can be included in nucleic acid molecules providedherein, such as 5′ UTRs of transcripts expressed in any cell or organ,including muscle, skin, subcutaneous tissue, liver, spleen, lymph nodes,antigen-presenting cells, and others. In another aspect, nucleic acidmolecules provided herein include a 5′ UTR comprising viral andnon-viral sequences. Accordingly, a 5′ UTR included in nucleic acidmolecules provided herein can comprise a combination of viral andnon-viral 5′ UTR sequences. In some aspects, the 5′ UTR included innucleic acid molecules provided herein is located upstream of or 5′ ofthe first polynucleotide that encodes one or more viral replicationproteins. In other aspects, the 5′ UTR is located 5′ of or upstream ofthe first polynucleotide of nucleic acid molecules provided herein thatencodes one or more viral replication proteins, and the firstpolynucleotide is located 5′ of or upstream of the second polynucleotideof nucleic acid molecules provided herein.

In one aspect, the 5′ UTR of nucleic acid molecules provided hereincomprises an alphavirus 5′ UTR. A 5′ UTR from any alphavirus can beincluded in nucleic acid molecules provided herein, including 5′ UTRsequences from Venezuelan Equine Encephalitis Virus (VEEV), EasternEquine Encephalitis Virus (EEEV), Everglades Virus (EVEV), Mucambo Virus(MUCV), Semliki Forest Virus (SFV), Pixuna Virus (PIXV), MiddleburgVirus (MIDV), Chikungunya Virus (CHIKV), O'Nyong-Nyong Virus (ONNV),Ross River Virus (RRV), Barmah Forest Virus (BFV), Getah Virus (GETV),Sagiyama Virus (SAGV), Bebaru Virus (BEBV), Mayaro Virus (MAYV), UnaVirus (UNAV), Sindbis Virus (SINV), Aura Virus (AURAV), Whataroa Virus(WHAV), Babanki Virus (BABV), Kyzylagach Virus (KYZV), Western EquineEncephalitis Virus (WEEV), Highland J Virus (HJV), Fort Morgan Virus(FMV), Ndumu Virus (NDUV), Salmonid Alphavirus (SAV), or Buggy CreekVirus (BCRV). In another aspect, the 5′ UTR comprises a sequence havingat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and anynumber or range in between, identity to a sequence of SEQ ID NO:73, SEQID NO:74, or SEQ ID NO:75. In yet another aspect, the 5′ UTR comprises asequence of SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75.

In some embodiments, the 5′ UTR comprises a sequence selected from the5′ UTRs of human IL-6, alanine aminotransferase 1, human apolipoproteinE, human fibrinogen alpha chain, human transthyretin, human haptoglobin,human alpha-1-antichymotrypsin, human antithrombin, humanalpha-1-antitrypsin, human albumin, human beta globin, human complementC3, human complement C5, SynK (thylakoid potassium channel proteinderived from the cyanobacteria, Synechocystis sp.), mouse beta globin,mouse albumin, and a tobacco etch virus, or fragments of any of theforegoing. Preferably, the 5′ UTR is derived from a tobacco etch virus(TEV). Preferably, an mRNA described herein comprises a 5′ UTR sequencethat is derived from a gene expressed by Arabidopsis thaliana.Preferably, the 5′ UTR sequence of a gene expressed by Arabidopsisthaliana is ATIG58420. Examples of 5 UTRs and 3′ UTRs are described inPCT/US2018/035419, the contents of which are herein incorporated byreference. Preferred 5′ UTR sequences comprise SEQ ID NOs: 5-10 and25-45: as shown in Table 1.

TABLE 1 5' UTR Sequences Name Sequence Seq ID No.: EVUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUC SEQ ID NO: 5AAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUU ACGAACGAUAG AT1G58420AUUAUUACAUCAAAACAAAAAGCCGCCA SEQ ID NO: 6 ARC5-2CUUAAGGGGGCGCUGCCUACGGAGGUGGCAGCCAUCUCCU SEQ ID NO: 7UCUCGGCAUCAAGCUUACCAUGGUGCCCCAGGCCCUGCUCUUGGUCCCGCUGCUGGUGUUCCCCCUCUGCUUCGGCAAGUUCCCCAUCUACACCAUCCCCGACAAGCUGGGGCCGUGGAGCCCCAUCGACAUCCACCACCUGUCCUGCCCCAACAACCUCGUGGUCGAGGACGAGGGCUGCACCAACCUGAGCGGGUUCUC CUAC HCVUGAGUGUCGU ACAGCCUCCA GGCCCCCCCC SEQ ID NO : 8 UCCCGGGAGA GCCAUAGUGGUCUGCGGAACCGGUGAGUAC ACCGGAAUUG CCGGGAAGAC UGGGUCCUUU CUUGGAUAAACCCACUCUAUGCCCGGCCAU UUGGGCGUGC CCCCGCAAGA CUGCUAGCCG AGUAGUGUUG GGUUGCGHUMAN AAUUAUUGGUUAAAGAAGUAUAUUAGUGCUAAUUUCCCU SEQ ID NO: 9 ALBUMINCCGUUUGUCCUAGCUUUUCUCUUCUGUCAACCCCACACGC CUUUGGCACA EMCVCUCCCUCCCC CCCCCCUAAC GUUACUGGCC SEQ ID NO: 10GAAGCCGCUU GGAAUAAGGC CGGUGUGCGU UUGUCUAUAU GUUAUUUUCC ACCAUAUUGCCGUCUUUUGG CAAUGUGAGG GCCCGGAAAC CUGGCCCUGU CUUCUUGACG AGCAUUCCUAGGGGUCUUUC CCCUCUCGCC AAAGGAAUGC AAGGUCUGUU GAAUGUCGUG AAGGAAGCAGUUCCUCUGGA AGCUUCUUGA AGACAAACAA CGUCUGUAGC GACCCUUUGC AGGCAGCGGAACCCCCCACC UGGCGACAGG UGCCUCUGCG GCCAAAAGCC ACGUGUAUAA GAUACACCUGCAAAGGCGGC ACAACCCCAG UGCCACGUUG UGAGUUGGAU AGUUGUGGAA AGAGUCAAAUGGCUCUCCUC AAGCGUAUUC AACAAGGGGC UGAAGGAUGC CCAGAAGGUA CCCCAUUGUAUGGGAUCUGA UCUGGGGCCU CGGUGCACAU GCUUUACGUG UGUUUAGUCG AGGUUAAAAAACGUCUAGGC CCCCCGAACC ACGGGGACGU GGUUUUCCUU UGAAAAACAC GAUGAUAAUAT1G67090 CACAAAGAGUAAAGAAGAACA SEQ ID NO: 25 AT1G35720AACACUAAAAGUAGAAGAAAA SEQ ID NO: 26 AT5G45900 CUCAGAAAGAUAAGAUCAGCCSEQ ID NO: 27 AT5G61250 AACCAAUCGAAAGAAACCAAA SEQ ID NO: 28 AT5G46430CUCUAAUCACCAGGAGUAAAA SEQ ID NO: 29 AT5G47110 GAGAGAGAUCUUAACAAAAAASEQ ID NO: 30 AT1G03110 UGUGUAACAACAACAACAACA SEQ ID NO: 31 AT3G12380CCGCAGUAGGAAGAGAAAGCC SEQ ID NO: 32 AT5G45910 AAAAAAAAAAGAAAUCAUAAASEQ ID NO: 33 AT1G07260 GAGAGAAGAAAGAAGAAGACG SEQ ID NO: 34 AT3G55500CAAUUAAAAAUACUUACCAAA SEQ ID NO: 35 AT3G46230 GCAAACAGAGUAAGCGAAACGSEQ ID NO: 36 AT2G36170 GCGAAGAAGACGAACGCAAAG SEQ ID NO: 37 AT1G10660UUAGGACUGUAUUGACUGGCC SEQ ID NO: 38 AT4G14340 AUCAUCGGAAUUCGGAAAAAGSEQ ID NO: 39 AT1G49310 AAAACAAAAGUUAAAGCAGAC SEQ ID NO: 40 AT4G14360UUUAUCUCAAAUAAGAAGGCA SEQ ID NO: 41 AT1G28520 GGUGGGGAGGUGAGAUUUCUUSEQ ID NO: 42 AT1G20160 UGAUUAGGAAACUACAAAGCC SEQ ID NO: 43 AT5G37370CAUUUUUCAAUUUCAUAAAAC SEQ ID NO: 44 AT4G11320 UUACUUUUAAGCCCAACAAAASEQ ID NO: 45 AT5G40850 GGCGUGUGUGUGUGUUGUUGA SEQ ID NO: 46 AT1G06150GUGGUGAAGGGGAAGGUUUAG SEQ ID NO: 47 AT2G26080 UUGUUUUUUUUUGGUUUGGUUSEQ ID NO: 48

3′ Untranslated Region (3′ UTR)

In some aspects, nucleic acid molecules provided herein further includea 3′ untranslated region (3′ UTR). Any 3′ UTR sequence can be includedin nucleic acid molecules provided herein. In one aspect, nucleic acidmolecules provided herein include a viral 3′ UTR. In another aspect,nucleic acid molecules provided herein include a non-viral 3′ UTR. Anynon-viral 3′ UTR can be included in nucleic acid molecules providedherein, such as 3′ UTRs of transcripts expressed in any cell or organ,including muscle, skin, subcutaneous tissue, liver, spleen, lymph nodes,antigen-presenting cells, and others. In some aspects, nucleic acidmolecules provided herein include a 3′ UTR comprising viral andnon-viral sequences. Accordingly, a 3′ UTR included in nucleic acidmolecules provided herein can comprise a combination of viral andnon-viral 3′ UTR sequences. In one aspect, the 3′ UTR is located 3′ ofor downstream of the second polynucleotide of nucleic acid moleculesprovided herein that comprises a first transgene encoding a firstantigenic protein or a fragment thereof. In another aspect, the 3′ UTRis located 3′ of or downstream of the second polynucleotide of nucleicacid molecules provided herein that comprises a first transgene encodinga first antigenic protein or a fragment thereof, and the secondpolynucleotide is located 3′ of or downstream of the firstpolynucleotide of nucleic acid molecules provided herein.

In one aspect, the 3′ UTR of nucleic acid molecules provided hereincomprises an alphavirus 3′ UTR. A 3′ UTR from any alphavirus can beincluded in nucleic acid molecules provided herein, including 3′ UTRsequences from Venezuelan Equine Encephalitis Virus (VEEV), EasternEquine Encephalitis Virus (EEEV), Everglades Virus (EVEV), Mucambo Virus(MUCV), Semliki Forest Virus (SFV), Pixuna Virus (PIXV), MiddleburgVirus (MIDV), Chikungunya Virus (CHIKV), O'Nyong-Nyong Virus (ONNV),Ross River Virus (RRV), Barmah Forest Virus (BFV), Getah Virus (GETV),Sagiyama Virus (SAGV), Bebaru Virus (BEBV), Mayaro Virus (MAYV), UnaVirus (UNAV), Sindbis Virus (SINV), Aura Virus (AURAV), Whataroa Virus(WHAV), Babanki Virus (BABV), Kyzylagach Virus (KYZV), Western EquineEncephalitis Virus (WEEV), Highland J Virus (HJV), Fort Morgan Virus(FMV), Ndumu Virus (NDUV), Salmonid Alphavirus (SAV), or Buggy CreekVirus (BCRV). In another aspect, the 3′ UTR comprises a sequence havingat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least %%, at least 97%, atleast 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and anynumber or range in between, identity to a sequence of SEQ ID NO:76. Inyet another aspect, the 3′ UTR comprises a poly-A sequence. In a furtheraspect, the 3′ UTR comprises a sequence of SEQ ID NO:76.

In some embodiments, the 3′ UTR comprises a sequence selected from the3′ UTRs of alanine aminotransferase 1, human apolipoprotein E, humanfibrinogen alpha chain, human haptoglobin, human antithrombin, humanalpha globin, human beta globin, human complement C₃, human growthfactor, human hepcidin, MALAT-1, mouse beta globin, mouse albumin, andXenopus beta globin, or fragments of any of the foregoing. In someembodiments, the 3′ UTR is derived from Xenopus beta globin. Exemplary3′ UTR sequences include SEQ ID NOs: 16-22 as shown in Table 2.

TABLE 2 3' UTR sequences. Name Sequence Seq ID No.: XBGCUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAG SEQ ID NO: 16CCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGU UUCUUCACAU HUMANUGCAAGGCUGGCCGGAAGCCCUUGCCUGAAAGCAAGA SEQ ID NO: 17 HAPTOGLOBINUUUCAGCCUGGAAGAGGGCAAAGUGGACGGGAGUGGACAGGAGUGGAUGCGAUAAGAUGUGGUUUGAAGCUGAUGGGUGCCAGCCCUGCAUUGCUGAGUCAAUCAAUAA AGAGCUUUCUUUUGACCCAU HUMANACGCCGAAGCCUGCAGCCAUGCGACCCCACGCCACCCC SEQ ID NO: 18 APOLIPO-GUGCCUCCUGCCUCCGCGCAGCCUGCAGCGGGAGACC PROTEINCUGUCCCCGCCCCAGCCGUCCUCCUGGGGUGGACCCU E AGUUUAAUAAAGAUUCACCAAGUUUCACGCAHCV UAGAGCGGCAAACCCUAGCUACACUCCAUAGCUAGUU SEQ ID NO: 19UCUUUUUUUUUUGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUCCUUUCUUUUCCUUCUUUUUUUCCUCUUUUCUUGGUGGCUCCAUCUUAGCCCUAGUCACGGCUAGCUGUGAAAGGUCCGUGAGCCGCAUGACUGCAGAGAGUGCCGUAACUGGUCUCUCUGCAGAUCAUGU MOUSEACACAUCACAACCACAACCUUCUCAGGCUACCCUGAG SEQ ID NO: 20 ALBUMINAAAAAAAGACAUGAAGACUCAGGACUCAUCUUUUCUGUUGGUGUAAAAUCAACACCCUAAGGAACACAAAUUUCUUUAAACAUUUGACUUCUUGUCUCUGUGCUGCAAUUA AUAAAAAAUGGAAAGAAUCUAC HUMAN ALPHAGCUGGAGCCUCGGUAGCCGUUCCUCCUGCCCGCUGGG SEQ ID NO: 21 GLOBINCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGGCCCUUCCUGGUCUUUGAAUAAAGUCUGAGUGGGCAGCA EMCVUAGUGCAGUCAC UGGCACAACG CGUUGCCCGG SEQ ID NO: 22 UAAGCCAAUC GGGUAUACACGGUCGUCAUACUGCAGACAG GGUUCUUCUA CUUUGCAAGA UAGUCUAGAG UAGUAAAAUAAAUAGUAUAAG

Triple Stop Codon

In some embodiments, the self-replicating RNA may comprise a sequenceimmediately downstream of a coding region (i.e., ORF) that creates atriple stop codon. A triple stop codon is a sequence of threeconsecutive stop codons. The triple stop codon can ensure totalinsulation of an expression cassette and may be incorporated to enhancethe efficiency of translation. In some embodiments, a self-replicatingRNA of the disclosure may comprise a triple combination of any of thesequences UAG, UGA, or UAA immediately downstream of a ORF describedherein. The triple combination can be three of the same codons, threedifferent codons, or any other permutation of the three stop codons.

Translation Enhancers and Kozak Sequences

For translation initiation, proper interactions between ribosomes andmRNAs must be established to determine the exact position of thetranslation initiation region. However, ribosomes also must dissociatefrom the translation initiation region to slide toward the downstreamsequence during mRNA translation. Translation enhancers upstream frominitiation sequences of mRNAs enhance the yields of proteinbiosynthesis. Several studies have investigated the effects oftranslation enhancers. In some embodiments, an mRNA described hereincomprises a translation enhancer sequence. These translation enhancersequences enhance the translation efficiency of a self-replicating RNAof the disclosure and thereby provide increased production of theprotein encoded by the mRNA. The translation enhancer region may belocated in the 5′ or 3′ UTR of an mRNA sequence. Examples of translationenhancer regions include naturally-occurring enhancer regions from theTEV 5′ UTR and the Xenopus beta-globin 3′ UTR. Exemplary 5′ UTR enhancersequences include but are not limited to those derived from mRNAsencoding human heat shock proteins (HSP) including HSP70-P2, HSP70-M1HSP72-M2, HSP17.9 and HSP70-P1. Preferred translation enhancer sequencesused in accordance with the embodiments of the present disclosure arerepresented by SEQ ID Nos: 11-15 as shown in Table 3.

TABLE 3 5' UTR Enhancers Seq ID Name Sequence No.: HSP70-GUCAGCUUUCAAACUCUUUGUUUCUUGUUU SEQ ID P2 GUUGAUUGAGAAUA NO: 11 HSP70-CUCUCGCCUGAGAAAAAAAAUCCACGAACC SEQ ID M1 AAUUUCUCAGCAACCAGCAGCACG NO: 12HISP72- ACCUGUGAGGGUUCGAAGGAAGUAGCAGUG SEQ ID M2 UUUUUUGUUCCUAGAGGAAGAGNO: 13 HSP17.9 ACACAGAAACAUUCGCAAAAACAAAAUCCC SEQ IDAGUAUCAAAAUUCUUCUCUUUUUUUCAUAU NO: 14 UUCGCAAAGAC HSP70-CAGAAAAAUUUGCUACAUUGUUUCACAAAC SEQ ID P1 UUCAAAUAUUAUUCAUUUAUUU NO: 15

In some embodiments, a self-replicating RNA of the disclosure comprisesa Kozak sequence. As is understood in the art, a Kozak sequence is ashort consensus sequence centered around the translational initiationsite of eukaryotic mRNAs that allows for efficient initiation oftranslation of the mRNA. See, for example, Kozak, Marilyn (1988) Mol.and Cell Biol, 8:2737-2744; Kozak, Marilyn (1991) J. Biol. Chem,266:19867-19870; Kozak, Marilyn (1990) Proc Natl. Acad. Sci. USA,87:8301-8305; and Kozak, Marilyn (1989) J. Cell Biol, 108:229-241. Itensures that a protein is correctly translated from the genetic message,mediating ribosome assembly and translation initiation. The ribosomaltranslation machinery recognizes the AUG initiation codon in the contextof the Kozak sequence. A Kozak sequence may be inserted upstream of thecoding sequence for the protein of interest, downstream of a 5′ UTR orinserted upstream of the coding sequence for the protein of interest anddownstream of a 5′ UTR. In some embodiments, a self-replicating RNAdescribed herein comprises a Kozak sequence having the amino acidsequence GCCACC (SEQ ID NO: 23). Preferably a self-replicating RNAdescribed herein comprises a partial Kozak sequence “p” having the aminoacid sequence GCCA (SEQ ID NO: 24).

Transgenes

Transgenes included in nucleic acid molecules provided herein can encodean antigenic protein or a fragment thereof. In some embodiments, secondpolynucleotides of nucleic acid molecules provided herein comprise afirst transgene. A first transgene included in second polynucleotides ofnucleic acid molecules provided herein can encode a first antigenicprotein or a fragment thereof. A transgene included in secondpolynucleotides of nucleic acid molecules provided herein can comprise asequence encoding the full amino acid sequence of an antigenic proteinor a sequence encoding any suitable portion or fragment of the fullamino acid sequence of an antigenic protein. In some embodiments, theantigenic protein is a coronavirus protein.

In another embodiment, the antigenic protein, when administered to amammalian subject, raises an immune response to a pathogen, such as acoronavirus. In some more particular embodiments, the antigenic proteinis expressed on the outer surface of the coronavirus; while in othermore particular embodiments, the antigen may be a non-surface antigen,e.g., useful as a T-cell epitope. The immunogen may elicit an immuneresponse against a coronavirus. The immune response may comprise anantibody response (usually including IgG) and/or a cell mediated immuneresponse. The polypeptide immunogen will typically elicit an immuneresponse that recognizes the corresponding coronavirus The immunogenwill typically be a surface polypeptide e.g. an envelope glycoprotein, aspike glycoprotein, etc.

In some aspects, the viral protein encoded by transgenes included innucleic acid molecules provided herein is a coronavirus protein. In someembodiments, the antigenic protein is a SARS-CoV-2 protein.

In one aspect, the antigenic protein is a SARS-CoV-2 spike glycoproteinor a fragment thereof. In another aspect, the SARS-CoV-2 spikeglycoprotein is a wild-type SARS-CoV-2 spike glycoprotein. In someaspects, the wild-type SARS-CoV-2 spike glycoprotein has an amino acidsequence of SEQ ID NO:123. In yet another aspect, the secondpolynucleotide of nucleic acid molecules provided herein comprises asequence having at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and anynumber or range in between, identity to a sequence of SEQ ID NO:121 orSEQ ID NO:122. In another aspect, the second polynucleotide of nucleicacid molecules provided herein comprises a sequence of SEQ ID NO:121 orSEQ ID NO:122. Accordingly, in some aspects, first transgenes includedin second polynucleotides of nucleic acid molecules provided hereincomprise a sequence having at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%,at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least99.9%, and any number or range in between, or 100% identity to asequence of SEQ ID NO:121 or SEQ ID NO:122.

In one aspect, the second polynucleotide of nucleic acid moleculesprovided herein encodes a wild-type SARS-CoV-2 spike glycoprotein or afragment thereof. In some aspects, a wild-type SARS-CoV-2 spikeglycoprotein comprises a sequence of SEQ ID NO:123. In another aspect,the second polynucleotide of nucleic acid molecules provided hereinencodes a SARS-CoV-2 spike protein comprising one or more mutations ascompared to a wild-type SARS-CoV-2 spike glycoprotein sequence.Mutations can include substitutions, deletions, insertions, and others.Mutations can be present at any position or at any combination ofpositions of a SARS-CoV-2 spike glycoprotein. Any number ofsubstitutions, insertions, deletions, or combinations thereof, can bepresent at any one or more positions of a SARS-CoV-2 spike glycoprotein.As an example, substitutions can include a change of a wild-type aminoacid at any position or at any combination of positions to any otheramino acid or combination of any other amino acids. Exemplary mutationsinclude mutations at positions 614, 936, 320, 477, 986, 987, or anycombination thereof. In one aspect, a SARS-CoV-2 spike glycoprotein or afragment thereof encoded by transgenes of second polynucleotidesincluded in nucleic acid molecules provided herein includes a D614Gmutation, a D936Y mutation, a D93611 mutation, a V320G mutation, anS477N mutation, an S4771 mutation, an S477T mutation, a K986P mutation,a V987P mutation, or any combination thereof. Additional mutations andvariants can be found in the National Bioinformatics Center 2019 NovelCoronavirus Information Database (2019nCoVR), National Genomics DataCenter, China National Center for Bioinformation/Beijing Institute ofGenomics, Chinese Academy of Science atbigd.big.ac.cn/ncov/variation/annotation. In another aspect, the secondpolynucleotide includes a transgene encoding a SARS-CoV-2 glycoproteinhaving at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, atleast 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and anynumber or range in between, or 100% identity to a sequence of SEQ IDNO:123.

In some aspects, the second polynucleotide of nucleic acid moleculesprovided herein comprises at least two transgenes, such as a secondcoronavirus protein. Any number of transgenes can be included in secondpolynucleotides of nucleic acid molecules provided herein, such as one,two, three, four, five, six, seven, eight, nine, ten, or moretransgenes. In one aspect, the second polynucleotide of nucleic acidmolecules provided herein includes a second transgene encoding a secondantigenic protein or a fragment thereof or an immunomodulatory protein.In one aspect, the second polynucleotide further comprises an internalribosomal entry site (IRES), a sequence encoding a 2A peptide, or acombination thereof, located between transgenes. As used herein, theterm “2A peptide” refers to a small (generally 18-22 amino acids)sequence that allows for efficient, stoichiometric production ofdiscrete protein products within a single reading frame through aribosomal skipping event within the 2A peptide sequence. As used herein,the term “internal ribosomal entry site” or “IRES” refers to anucleotide sequence that allows for the initiation of proteintranslation of a messenger RNA (mRNA) sequence in the absence of an AUGstart codon or without using an AUG start codon. An IRES can be foundanywhere in an mRNA sequence, such as at or near the beginning, at ornear the middle, or at or near the end of the mRNA sequence, forexample.

Any number of transgenes included in second polynucleotides of nucleicacid molecules provided herein can be expressed via any combination of2A peptide and IRES sequences. For example, a second transgene located3′ of a first transgene can be expressed via a 2A peptide sequence orvia an IRES sequence. As another example, a second transgene located 3′of a first transgene and a third transgene located 3′ of the secondtransgene can be expressed via 2A peptide sequences located between thefirst and second transgenes and the second and third transgenes, via anIRES sequence located between the first and second transgenes and thesecond and third transgenes, via a 2A peptide sequence located betweenthe first and second transgenes and an IRES located between the secondand third transgenes, or via an IRES sequence located between the firstand second transgenes and a 2A peptide sequence located between thesecond and third transgenes. Similar configurations and combinations of2A peptide and IRES sequences located between transgenes arecontemplated for any number of transgenes included in secondpolynucleotides of nucleic acid molecules provided herein. In additionto expression via 2A peptide and IRES sequences, two or more transgenesincluded in nucleic acid molecules provided herein can also be expressedfrom separate subgenomic RNAs.

A second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth,etc., transgene included in second polynucleotides of nucleic acidmolecules provided herein can encode an immunomodulatory protein or afunctional fragment or functional variant thereof. Any immunomodulatoryprotein or a functional fragment or functional variant thereof can beencoded by a transgene included in second polynucleotides.

As used herein, the terms “functional variant” or “functional fragment”refer to a molecule, including a nucleic acid or protein, for example,that comprises a nucleotide and/or amino acid sequence that is alteredby one or more nucleotides and/or amino acids compared to the nucleotideand/or amino acid sequences of the parent or reference molecule. For aprotein, a functional variant is still able to function in a manner thatis similar to the parent molecule. In other words, the modifications inthe amino acid and/or nucleotide sequence of the parent molecule do notsignificantly affect or alter the functional characteristics of themolecule encoded by the nucleotide sequence or containing the amino acidsequence. The functional variant may have conservative sequencemodifications including nucleotide and amino acid substitutions,additions and deletions. These modifications can be introduced bystandard techniques known in the art, such as site-directed mutagenesisand random PCR-mediated mutagenesis. Functional variants can alsoinclude, but are not limited to, derivatives that are substantiallysimilar in primary structural sequence, but which contain, e.g., invitro or in vivo modifications, chemical and/or biochemical, that arenot found in the parent molecule. Such modifications include, interalia, acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA-mediated addition of amino acidsto proteins such as arginylation, ubiquitination, and the like.

In one aspect, a second transgene included in second polynucleotides ofnucleic acid molecules provided herein encodes a cytokine, a chemokine,or an interleukin. Exemplary cytokines include interferons, TNF-α,TGF-β, G-CSF, and GM-CSF. Exemplary chemokines include CCL3, CCL26, andCXCL7. Exemplary interleukins include IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, and IL-23. Anytransgene or combination of transgenes encoding any cytokine, chemokine,interleukin, or combinations thereof, can be included in secondpolynucleotides of nucleic acid molecules provided herein.

In some embodiments, the second transgene encodes a second coronavirusprotein.

DNA and RNA Molecules

Nucleic acid molecules provided herein can be DNA molecules or RNAmolecules. It will be appreciated that T present in DNA is substitutedwith U in RNA, and vice versa. In one aspect, nucleic acid moleculesprovided herein are DNA molecules. In another aspect, DNA moleculesprovided herein further comprise a promoter. As used herein, the term“promoter” refers to a regulatory sequence that initiates transcription.A promoter can be operably linked to first and second polynucleotides ofnucleic acid molecules provided herein. Generally, promoters included inDNA molecules provided herein include promoters for in vitrotranscription (IVT). Any suitable promoter for in vitro transcriptioncan be included in DNA molecules provided herein, such as a T7 promoter,a T3 promoter, an SP6 promoter, and others. In one aspect, DNA moleculesprovided herein comprise a T7 promoter. In another aspect, the promoteris located 5′ of the 5′ UTR included in DNA molecules provided herein.In yet another aspect, the promoter is a T7 promoter located 5′ of the5′ UTR included in DNA molecules provided herein. In yet another aspect,the promoter overlaps with the 5′ UTR. A promoter and a 5′ UTR canoverlap by about one nucleotide, about two nucleotides, about threenucleotides, about four nucleotides, about five nucleotides, about sixnucleotides, about seven nucleotides, about eight nucleotides, aboutnine nucleotides, about ten nucleotides, about 11 nucleotides, about 12nucleotides, about 13 nucleotides, about 14 nucleotides, about 15nucleotides, about 16 nucleotides, about 17 nucleotides, about 18nucleotides, about 19 nucleotides, about 20 nucleotides, about 21nucleotides, about 22 nucleotides, about 23 nucleotides, about 24nucleotides, about 25 nucleotides, about 26 nucleotides, about 27nucleotides, about 28 nucleotides, about 29 nucleotides, about 30nucleotides, about 31 nucleotides, about 32 nucleotides, about 33nucleotides, about 34 nucleotides, about 35 nucleotides, about 36nucleotides, about 37 nucleotides, about 38 nucleotides, about 39nucleotides, about 40 nucleotides, about 41 nucleotides, about 42nucleotides, about 43 nucleotides, about 44 nucleotides, about 45nucleotides, about 46 nucleotides, about 47 nucleotides, about 48nucleotides, about 49 nucleotides, about 50 nucleotides, or morenucleotides.

In some aspects, DNA molecules provided herein include a promoter for invivo transcription. Generally, the promoter for in vivo transcription isan RNA polymerase II (RNA pol II) promoter. Any RNA pol II promoter canbe included in DNA molecules provided herein, including constitutivepromoters, inducible promoters, and tissue-specific promoters. Exemplaryconstitutive promoters include a cytomegalovirus (CMV) promoter, an EF1αpromoter, an SV40 promoter, a PGK1 promoter, a Ubc promoter, a humanbeta actin promoter, a CAG promoter, and others. Any tissue-specificpromoter can be included in DNA molecules provided herein. In oneaspect, the RNA pol II promoter is a muscle-specific promoter,skin-specific promoter, subcutaneous tissue-specific promoter,liver-specific promoter, spleen-specific promoter, lymph node-specificpromoter, or a promoter with any other tissue specificity. DNA moleculesprovided herein can also include an enhancer. Any enhancer thatincreases transcription can be included in DNA molecules providedherein.

In some aspects, nucleic acid molecules provided herein are RNAmolecules. An RNA molecule provided herein can be generated by in vitrotranscription (IVT) of DNA molecules provided herein. In one aspect, RNAmolecules provided herein are self-replicating RNA molecules. In anotheraspect, RNA molecules provided herein further comprise a 5′ cap. Any 5′cap can be included in RNA molecules provided herein, including 5′ capshaving a Cap 1 structure, a Cap 1 (m6A) structure, a Cap 2 structure, aCap 0 structure, or any combination thereof. In one aspect, RNAmolecules provided herein include a 5′ cap having Cap 1 structure. Inyet another aspect, RNA molecules provided herein are self-replicatingRNA molecules comprising a 5′ cap having a Cap 1 structure. In a furtheraspect, RNA molecules provided herein comprise a cap having a Cap 1structure, wherein a m7G is linked via a 5′-5′ triphosphate to the 5′end of the 5′ UTR. In yet a further aspect, RNA molecules providedherein comprise a cap having a Cap 1 structure, wherein a m7G is linkedvia a 5′-5′ triphosphate to the 5′ end of the 5′ UTR comprising asequence of SEQ ID NO:73. Any method of capping can be used, including,but not limited to using a Vaccinia Capping enzyme (New England Biolabs,Ipswich, Mass.) and co-transcriptional capping or capping at or shortlyafter initiation of in vitro transcription (IVT), by for example,including a capping agent as part of an in vitro transcription (IVT)reaction. (Nuc. Acids Symp. (2009) 53:129).

Provided herein, in some embodiments, are nucleic acid moleculescomprising (a) a sequence of SEQ ID NO:10; (b) a sequence of SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:76, and SEQ ID NO:77, wherein T issubstituted with U; (c) a sequence of SEQ ID NO:124; (d) a sequence ofSEQ ID NO:124, wherein T is substituted with U; (e) a sequence of SEQ IDNO:125; or (f) a sequence of SEQ ID NO:125, wherein T is substitutedwith U. In one aspect, nucleic acid molecules provided herein are RNAmolecules. In another aspect, RNA molecules provided herein furthercomprise a 5′ cap having a Cap 1 structure. Any RNA molecules providedherein can be self-replicating RNA molecules.

Only those mRNAs that carry the Cap structure are active in Capdependent translation; “decapitation” of mRNA results in an almostcomplete loss of their template activity for protein synthesis (Nature,255:33-37, (1975); J. Biol. Chem., vol. 253:5228-5231, (1978); and Proc.Natl. Acad. Sci. USA, 72:1189-1193, (1975)).

Another element of eukaryotic mRNA is the presence of 2′-O-methylnucleoside residues at transcript position 1 (Cap 1), and in some cases,at transcript positions 1 and 2 (Cap 2). The 2′-O-methylation of mRNAprovides higher efficacy of mRNA translation in vivo (Proc. Natl. Acad.Sci. USA, 77:3952-3956 (1980)) and further improves nuclease stabilityof the 5′-capped mRNA. The mRNA with Cap 1 (and Cap 2) is a distinctivemark that allows cells to recognize the bona fide mRNA 5′ end, and insome instances, to discriminate against transcripts emanating frominfectious genetic elements (Nucleic Acid Research 43: 482-492 (2015)).

Some examples of 5′ cap structures and methods for preparing mRNAscomprising the same are given in WO2015/051169A2, WO/2015/061491, US2018/0273576, and U.S. Pat. Nos. 8,093,367, 8,304,529, and 10,487,105.In some embodiments, the 5′ cap is m7GpppAmpG, which is known in theart. In some embodiments, the 5′ cap is m7GpppG or m7GpppGm, which areknown in the art. Structural formulas for embodiments of 5′ capstructures are provided below.

In some embodiments, a self-replicating RNA of the disclosure comprisesa 5′ cap having the structure of Formula (Cap I).

wherein B¹ is a natural or modified nucleobase; R¹ and R² are eachindependently selected from a halogen, OH, and OCH₃; each L isindependently selected from the group consisting of phosphate,phophorothioate, and boranophosphate wherein each L is linked by diesterbonds; n is 0 or 1. and mRNA represents an mRNA of the presentdisclosure linked at its 5′ end. In some embodiments B¹ is G, m⁷G, or A.In some embodiments, n is 0. In some embodiments n is 1. In someembodiments, B¹ is A or m⁷A and R¹ is OCH₃; wherein G is guanine, m⁷G is7-methylguanine, A is adenine, and m⁶A is N⁶-methyladenine.

In some embodiments, a self-replicating RNA of the disclosure comprisesa 5′ cap having the structure of Formula (Cap II).

wherein B¹ and B² are each independently a natural or modifiednucleobase; R¹, R², and R³ are each independently selected from ahalogen, OH, and OCH₃; each L is independently selected from the groupconsisting of phosphate, phophorothioate, and boranophosphate whereineach L is linked by diester bonds; mRNA represents an mRNA of thepresent disclosure linked at its 5′ end; and n is 0 or 1. In someembodiments B¹ is G, m⁷G, or A. In some embodiments, n is 0. In someembodiments, n is 1. In some embodiments, B¹ is A or m⁶A and R¹ is OCH₃;wherein G is guanine, m⁷G is 7-methylguanine, A is adenine, and m⁷A isN⁶-methyladenine.

In some embodiments, a self-replicating RNA of the disclosure comprisesa 5′ cap having the structure of Formula (Cap III).

wherein B1, B2, and B3 are each independently a natural or modifiednucleobase; R¹, R², R³, and R⁴ are each independently selected from ahalogen, OH, and OCH₃; each L is independently selected from the groupconsisting of phosphate, phosphorothioate, and boranophosphate whereineach L is linked by diester bonds; mRNA represents an mRNA of thepresent disclosure linked at its 5′ end; and n is 0 or 1. In someembodiments, at least one of R¹, R², R³, and R⁴ is OH. In someembodiments B1 is G, m7G, or A. In some embodiments, B1 is A or m6A andR1 is OCH₃; wherein G is guanine, m7G is 7-methylguanine, A is adenine,and m6A is N6-methyladenine. In some embodiments, n is 1.

In some embodiments, a self-replicating RNA of the disclosure comprisesa m7GpppG 5′ cap analog having the structure of Formula (Cap IV).

wherein, R¹, R², and R³ are each independently selected from a halogen,OH, and OCH₃; each L is independently selected from the group consistingof phosphate, phosphorothioate, and boranophosphate wherein each L islinked by diester bonds; mRNA represents an mRNA of the presentdisclosure linked at its 5′ end; n is 0 or 1. In some embodiments, atleast one of R¹, R², and R³ is OH. In some embodiments, the 5′ cap ism⁷GpppG wherein R¹, R², and R³ are each OH, n is 1, and each L is aphosphate. In some embodiments, n is 1. In some embodiments, the 5′ capis m7GpppGm, wherein R¹ and R² are each OH, R³ is OCH₃, each L is aphosphate, mRNA is the mRNA encoding an enzyme having OTC activitylinked at its 5′ end, and n is 1.

In some embodiments, a self-replicating RNA of the disclosure comprisesa m7Gpppm7G 5′ cap analog having the structure of Formula (Cap V).

wherein, R¹, R², and R³ are each independently selected from a halogen,OH, and OCH₃; each L is independently selected from the group consistingof phosphate, phosphorothioate, and boranophosphate wherein each L islinked by diester bonds; mRNA represents an mRNA of the presentdisclosure linked at its 5′ end; and n is 0 or 1. In some embodiments,at least one of R¹, R², and R³ is OH. In some embodiments, n is 1.

In some embodiments, a self-replicating RNA of the disclosure comprisesa m7Gpppm7GpN, 5′ cap analog, wherein N is a natural or modifiednucleotide, the 5′ cap analog having the structure of Formula (Cap VI).

wherein B3 is a natural or modified nucleobase; R¹, R², R³, and R⁴ areeach independently selected from a halogen, OH, and OCH₃; each L isindependently selected from the group consisting of phosphate,phosphorothioate, and boranophosphate wherein each L is linked bydiester bonds; mRNA represents an mRNA of the present disclosure linkedat its 5′ end; and n is 0 or 3. In some embodiments, at least one of R¹,R², R³, and R⁴ is OH. In some embodiments B¹ is G, m⁷G, or A. In someembodiments, B¹ is A or m⁶A and R¹ is OCH₃; wherein G is guanine, m⁷G is7-methylguanine, A is adenine, and m⁶A is N⁶-methyladenine. In someembodiments, n is 1.

In some embodiments, a self-replicating RNA of the disclosure comprisesa m7Gpppm7GpG 5′ cap analog having the structure of Formula (Cap VII).

wherein, R¹, R², R³, and R⁴ are each independently selected from ahalogen, OH, and OCH₃; each L is independently selected from the groupconsisting of phosphate, phosphorothioate, and boranophosphate whereineach L is linked by diester bonds; mRNA represents an mRNA of thepresent disclosure linked at its 5′ end; and n is 0 or 1. In someembodiments, at least one of R¹, R², R³, and R⁴ is OH. In someembodiments, n is 1.

In some embodiments, a self-replicating RNA of the disclosure comprisesa m7Gpppm7Gpm7G 5′ cap analog having the structure of Formula (CapVIII).

wherein, R¹, R², R³, and R⁴ are each independently selected from ahalogen, OH, and OCH₃; each L is independently selected from the groupconsisting of phosphate, phosphorothioate, and boranophosphate whereineach L is linked by diester bonds; mRNA represents an mRNA of thepresent disclosure linked at its 5′ end; n is 0 or 1. In someembodiments, at least one of R¹, R², R³, and R⁴ is OH. In someembodiments, n is 1.

In some embodiments, a self-replicating RNA of the disclosure comprisesa m7GpppA 5′ cap analog having the structure of Formula (Cap IX).

wherein, R¹, R², and R³ are each independently selected from a halogen,OH, and OCH₃; each L is independently selected from the group consistingof phosphate, phosphorothioate, and boranophosphate wherein each L islinked by diester bonds; mRNA represents an mRNA of the presentdisclosure linked at its 5′ end; and n is 0 or 1. In some embodiments,at least one of R¹, R², and R³ is OH. In some embodiments, n is 1.

In some embodiments, a self-replicating RNA of the disclosure comprisesa m7GpppApN 5′ cap analog, wherein N is a natural or modifiednucleotide, and the 5′ cap has the structure of Formula (Cap X).

wherein B³ is a natural or modified nucleobase; R¹, R², R³, and R areeach independently selected from a halogen, OH, and OCH₃; each L isindependently selected from the group consisting of phosphate,phosphorothioate, and boranophosphate wherein each L is linked bydiester bonds; mRNA represents an mRNA of the present disclosure linkedat its 5′ end; and n is 0 or 1. In some embodiments, at least one of R¹,R², R³, and R is OH. In some embodiments B³ is G, m⁷G, A or m⁶A; whereinG is guanine, m⁷G is 7-methylguanine, A is adenine, and m⁶A isN⁶-methyladenine. In some embodiments, n is 1.

In some embodiments, a self-replicating RNA of the disclosure comprisesa

wherein, R¹, R², and R⁴ are each independently selected from a halogen,OH, and OCH₃, each L is independently selected from the group consistingof phosphate, phosphorothioate, and boranophosphate wherein each L islinked by diester bonds; mRNA represents an mRNA of the presentdisclosure linked at its 5′ end; and n is 0 or 1. In some embodiments,at least one of R¹, R², and R⁴ is OH. In some embodiments, the compoundof Formula Cap XI is m⁷GpppAmpG, wherein R¹, R², and R⁴ are each OH, nis 1, and each L is a phosphate linkage. In some embodiments, n is 1.

In some embodiments, a self-replicating RNA of the disclosure comprisesa m7GpppApm7G 5′ cap analog having the structure of Formula (Cap XII).

wherein, R¹, R², R³, and R⁴ are each independently selected from ahalogen, OH, and OCH₃; each L is independently selected from the groupconsisting of phosphate, phosphorothioate, and boranophosphate whereineach L is linked by diester bonds; mRNA represents an mRNA of thepresent disclosure linked at its 5′ end; and n is 0 or 1. In someembodiments, at least one of R¹, R², R³, and R⁴ is OH. In someembodiments, n is 1.

In some embodiments, a self-replicating RNA of the disclosure comprisesa m7GpppApm7G 5′ cap analog having the structure of Formula (Cap XIII).

wherein, R¹, R², and R⁴ are each independently selected from a halogen,OH, and OCH₃; each L is independently selected from the group consistingof phosphate, phosphorothioate, and boranophosphate wherein each L islinked by diester bonds; mRNA represents an mRNA of the presentdisclosure linked at its 5′ end; and n is 0 or 1. In some embodiments,at least one of R¹, R², and R⁴ is OH. In some embodiments, n is 1.

Poly-Adenine (Poly-A) Tail

Polyadenylation is the addition of a poly(A) tail, a chain of adeninenucleotides usually about 100-120 monomers in length, to mRNA. Ineukaryotes, polyadenylation is part of the process that produces maturemRNA for translation and begins as the transcription of a geneterminates. The 3′-most segment of a newly made pre-mRNA is firstcleaved off by a set of proteins; these proteins then synthesize thepoly(A) tail at the 3′ end. The poly(A) tail is important for thenuclear export, translation, and stability of mRNA. The tail isshortened over time, and, when it is short enough, the mRNA isenzymatically degraded. However, in a few cell types, mRNAs with shortpoly(A) tails are stored for later activation by re-polyadenylation inthe cytosol.

Preferably, a self-replicating RNA of the disclosure comprises a 3′ tailregion, which can serve to protect the RNA from exonuclease degradation.The tail region may be a 3′poly(A) and/or 3′poly(C) region. Preferably,the tail region is a 3′ poly(A) tail. As used herein a “3′ poly(A) tail”is a polymer of sequential adenine nucleotides that can range in sizefrom, for example: 10 to 250 sequential adenine nucleotides; 60-125sequential adenine nucleotides, 90-125 sequential adenine nucleotides,95-125 sequential adenine nucleotides, 95-121 sequential adeninenucleotides, 100 to 121 sequential adenine nucleotides, 110-121sequential adenine nucleotides; 112-121 sequential adenine nucleotides;114-121 adenine sequential nucleotides; or 115 to 121 sequential adeninenucleotides. Preferably, a 3′ poly(A) tail as described herein comprise90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, or 125 sequential adenine nucleotides. 3′Poly(A) tails can be added using a variety of methods known in the art,e.g., using poly(A) polymerase to add tails to synthetic or in vitrotranscribed RNA. Other methods include the use of a transcription vectorto encode poly(A) tails or the use of a ligase (e.g., via splintligation using a T4 RNA ligase and/or T4 DNA ligase), wherein poly(A)may be ligated to the 3 end of a sense RNA. In some embodiments, acombination of any of the above methods is utilized.

Design and Synthesis of Self-Replicating RNA

The constructs for exemplary self-replicating RNA sequences of thepresent disclosure are provided in Tables 4-5.

TABLE 4 Comparison of STARR™ self-replicatingRNA of the disclosure with comparative self-replicating RNA as describedSequence Construct Position Type Sequence STARR™ 5' UTR nucleotideATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCT (SEQ ID ACCCAAA NO: 49) STARR™non- nucleotide ATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCC (SEQ ID structuralCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTT NO: 50) gene ORFGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCOATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCCGTCGACGGCCCCACCAGCCTGTACCACCAGGCCAACAAGGGCGTGAGGGTGGCCTACTGGATCGGCTTCGACACCACACCCTTCATGTTCAAGAACCTGGCCGGCGCCTACCCCAGCTACAGCACCAACTGGGCCGACGAGACCGTGCTGACCGCCAGGAACATCGGCCTGTGCAGCAGCGACGTGATGGAGAGGAGCCGGAGAGGCATGAGCATCCTGAGGAAGAAATACCTGAAGCCCAGCAACAACGTGCTGTTCAGCGTGGGCAGCACCATCTACCACGAGAAGAGGGACCTGCTCAGGAGCTGGCACCTGCCCAGCGTGTTCCACCTGAGGGGCAAGCAGAACTACACCTGCAGGTGCGAGACCATCGTGAGCTGCGACGGCTACGTGGTGAAGAGGATCGCCATCAGCCCCGGCCTGTACGGCAAGCCCAGCGGCTACGCCGCTACAATGCACAGGGAGGGCTTCCTGTGCTGCAAGGTGACCGACACCCTGAACGGCGAGAGGGTGAGCTTCCCCGTGTGCACCTACGTGCCCGCCACCCTGTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGAGCGCCGACGACGCCCAGAAGCTGCTCGTGGGCCTGAACCAGAGGATCGTGGTCAACGGCAGGACCCAGAGGAACACCAACACAATGAAGAACTACCTGCTGCCCGTGGTGGCCCAGGCTTTCGCCAGGTGGGCCAAGGAGTACAAGGAGGACCAGGAAGACGAGAGGCCCCTGGGCCTGAGGGACAGGCAGCTGGTGATGGGCTGCTGCTGGGCCTTCAGGCGGCACAAGATCACCAGCATCTACAAGAGGCCCGACACCCAGACCATCATCAAGGTGAACAGCGACTTCCACAGCTTCGTGCTGCCCAGGATCGGCAGCAACACCCTGGAGATCGGCCTGAGGACCCGGATCAGGAAGATGCTGGAGGAACACAAGGAGCCCAGCCCACTGATCACCGCCGAGGACGTGCAGGAGGCCAAGTGCGCTGCCGACGAGGCCAAGGAGGTGAGGGAGGCCGAGGAACTGAGGGCCGCCCTGCCACCCCTGGCTGCCGACGTGGAGGAACCCACCCTGGAAGCCGACGTGGACCTGATGCTGCAGGAGGCCGGCGCCGGAAGCGTGGAGACACCCAGGGGCCTGATCAAGGTGACCAGCTACGACGGCGAGGACAAGATCGGCAGCTACGCCGTGCTGAGCCCACAGGCCGTGCTGAAGTCCGAGAAGCTGAGCTGCATCCACCCACTGGCCGAGCAGGTGATCGTGATCACCCACAGCGGCAGGAAGGGCAGGTACGCCGTGGAGCCCTACCACGGCAAGGTGGTCGTGCCCGAGGGCCACGCCATCCCCGTGCAGGACTTCCAGGCCCTGAGCGAGAGCGCCACCATCGTGTACAACGAGAGGGAGTTCGTGAACAGGTACCTGCACCATATCGCCACCCACGGCGGAGCCCTGAACACCGACGAGGAATACTACAAGACCGTGAAGCCCAGCGAGCACGACGGCGAGTACCTGTACGACATCGACAGGAAGCAGTGCGTGAAGAAAGAGCTGGTGACCGGCCTGGGACTGACCGGCGAGCTGGTGGACCCACCCTTCCACGAGTTCGCCTACGAGAGCCTGAGGACCAGACCCGCCGCTCCCTACCAGGTGCCCACCATCGGCGTGTACGGCGTGCCCGGCAGCGGAAAGAGCGGCATCATCAAGAGCGCCGTGACCAAGAAAGACCTGGTGGTCAGCGCCAAGAAAGAGAACTGCGCCGAGATCATCAGGGACGTGAAGAAGATGAAAGGCCTGGACGTGAACGCGCGCACCGTGGACAGCGTGCTGCTGAACGGCTGCAAGCACCCCGTGGAGACCCTGTACATCGACGAGGCCTTCGCTTGCCACGCCGGCACCCTGAGGGCCCTGATCGCCATCATCAGGCCCAAGAAAGCCGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATGATGTGCCTGAAGGTGCACTTCAACCACGAGATCTGCACCCAGGTGTTCCACAAGAGCATCAGCAGGCGGTGCACCAAGAGCGTGACCAGCGTCGTGAGCACCCTGTTCTACGACAAGAAAATGAGGACCACCAACCCCAAGGAGACCAAAATCGTGATCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCTGATCCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCAGATCGACTACAAGGGCAACGAGATCATGACCGCCGCTGCCAGCCAGGGCCTGACCAGGAAGGGCGTGTACGCCGTGAGGTACAAGGTGAACGAGAACCCACTGTACGCTCCCACCAGCGAGCACGTGAACGTGCTGCTGACCAGGACCGAGGACAGGATCGTGTGGAAGACCCTGGCCGGCGACCCCTGGATCAAGACCCTGACCGCCAAGTACCCCGGCAACTTCACCGCCACCATCGAAGAGTGGCAGGCCGAGCACGACGCCATCATGAGGCACATCCTGGAGAGGCCCGACCCCACCGACGTGTTCCAGAACAAGGCCAACGTGTGCTGGGCCAAGGCCCTGGTGCCCGTGCTGAAGACCGCCGGCATCGACATGACCACAGAGCAGTGGAACACCGTGGACTACTTCGAGACCGACAAGGCCCACAGCGCCGAGATCGTGCTGAACCAGCTGTGCGTGAGGTTCTTCGGCCTGGACCTGGACAGCGGCCTGTTCAGCGCCCCCACCGTGCCACTGAGCATCAGGAACAACCACTGGGACAACAGCCCCAGCCCAAACATGTACGGCCTGAACAAGGAGGTGGTCAGGCAGCTGAGCAGGCGGTACCCACAGCTGCCCAGGGCCGTGGCCACCGGCAGGGTGTACGACATGAACACCGGCACCCTGAGGAACTACGACCCCAGGATCAACCTGGTGCCCGTGAACAGGCGGCTGCCCCACGCCCTGGTGCTGCACCACAACGAGCACCCACAGAGCGACTTCAGCTCCTTCGTGAGCAAGCTGAAAGGCAGGACCGTGCTGGTCGTGGGCGAGAAGCTGAGCGTGCCCGGCAAGATGGTGGACTGGCTGAGCGACAGGCCCGAGGCCACCTTCCGGGCCAGGCTGGACCTCGGCATCCCCGGCGACGTGCCCAAGTACGACATCATCTTCGTGAACGTCAGGACCCCATACAAGTACCACCATTACCAGCAGTGCGAGGACCACGCCATCAAGCTGAGCATGCTGACCAAGAAGGCCTGCCTGCACCTGAACCCCGGAGGCACCTGCGTGAGCATCGGCTACGGCTACGCCGACAGGGCCAGCGAGAGCATCATTGGCGCCATCGCCAGGCTGTTCAAGTTCAGCAGGGTGTGCAAACCCAAGAGCAGCCTGGAGGAAACCGAGGTGCTGTTCGTGTTCATCGGCTACGACCGGAAGGCCAGGACCCACAACCCCTACAAGCTGAGCAGCACCCTGACAAACATCTACACCGGCAGCAGGCTGCACGAGGCCGGCTGCGCCCCCAGCTACCACGTGGTCAGGGGCGATATCGCCACCGCCACCGAGGGCGTGATCATCAACGCTGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGTGTGCGGCGCCCTGTACAAGAAGTTCCCCGAGAGCTTCGACCTGCAGCCCATCGAGGTGGGCAAGGCCAGGCTGGTGAAGGGCGCCGCTAAGCACATCATCCACGCCGTGGGCCCCAACTTCAACAAGGTGAGCGAGGTGGAAGGCGACAAGCAGCTGGCCGAAGCCTACGAGAGCATCGCCAAGATCGTGAACGACAATAACTACAAGAGCGTGGCCATCCCACTGCTCAGCACCGGCATCTTCAGCGGCAACAAGGACAGGCTGACCCAGAGCCTGAACCACCTGCTCACCGCCCTGGACACCACCGATGCCGACGTGGCCATCTACTGCAGGGACAAGAAGTGGGAGATGACCCTGAAGGAGGCCGTGGCCAGGCGGGAGGCCGTGGAAGAGATCTGCATCAGCGACGACTCCAGCGTGACCGAGCCCGACGCCGAGCTGGTGAGGGTGCACCCCAAGAGCTCCCTGGCCGGCAGGAAGGGCTACAGCACCAGCGACGGCAAGACCTTCAGCTACCTGGAGGGCACCAAGTTCCACCAGGCCGCTAAGGACATCGCCGAGATCAACGCTATGTGGCCCGTGGCCACCGAGGCCAACGAGCAGGTGTGCATGTACATCCTGGGCGAGAGCATGTCCAGCATCAGGAGCAAGTGCCCCGTGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCTGCCCTGCCTGTGCATCCACGCTATGACACCCGAGAGGGTGCAGCGGCTGAAGGCCAGCAGGCCCGAGCAGATCACCGTGTGCAGCTCCTTCCCACTGCCCAAGTACAGGATCACCGGCGTGCAGAAGATCCAGTGCAGCCAGCCCATCCTGTTCAGCCCAAAGGTGCCCGCCTACATCCACCCCAGGAAGTACCTGGTGGAGACCCCACCCGTGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCTGATCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCATCATTATCGAGGAAGAGGAAGAGGACAGCATCAGCCTGCTGAGCGACGGCCCCACCCACCAGGTGCTGCAGGTGGAGGCCGACATCCACGGCCCACCCAGCGTGTCCAGCTCCAGCTGGAGCATCCCACACGCCAGCGACTTCGACGTGGACAGCCTGAGCATCCTGGACACCCTGGAGGGCGCCAGCGTGACCTCCGGCGCCACCAGCGCCGAGACCAACAGCTACTTCGCCAAGAGCATGGAGTTCCTGGCCAGGCCCGTGCCAGCTCCCAGGACCGTGTTCAGGAACCCACCCCACCCAGCTCCCAGGACCAGGACCCCAAGCCTGGCTCCCAGCAGGGCCTGCAGCAGGACCAGCCTGGTGAGCACCCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAACTGGAGGCCCTGACACCCAGCAGGACCCCCAGCAGGTCCGTGAGCAGGACTAGTCTGGTGTCCAACCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAATTCGAGGCCTTCGTGGCCCAGCAACAGAGACGGTTCGACGCCGGCGCCTACATCTTCAGCAGCGACACCGGCCAGGGACACCTGCAGCAAAAGAGCGTGAGGCAGACCGTGCTGAGCGAGGTGGTGCTGGAGAGGACCGAGCTGGAAATCAGCTACGCCCCCAGGCTGGACCAGGAGAAGGAGGAACTGCTCAGGAAGAAACTGCAGCTGAACCCCACCCCAGCCAACAGGAGCAGGTACCAGAGCAGGAAGGTGGAGAACATGAAGGCCATCACCGCCAGGCGGATCCTGCAGGGCCTGGGACACTACCTGAAGGCCGAGGGCAAGGTGGAGTGCTACAGGACCCTGCACCCCGTGCCACTGTACAGCTCCAGCGTGAACAGGGCCTTCTCCAGCCCCAAGGTGGCCGTGGAGGCCTGCAACGCTATGCTGAAGGAGAACTTCCCCACCGTGGCCAGCTACTGCATCATCCCCGAGTACGACGCCTACCTGGACATGGTGGACGGCGCCAGCTGCTGCCTGGACACCGCCAGCTTCTGCCCCGCCAAGCTGAGGAGCTTCCCCAAGAAACACAGCTACCTGGAGCCCACCATCAGGAGCGCCGTGCCCAGCGCCATCCAGAACACCCTGCAGAACGTGCTGGCCGCTGCCACCAAGAGGAACTGCAACGTGACCCAGATGAGGGAGCTGCCCGTGCTGGACAGCGCTGCCTTCAACGTGGAGTGCTTCAAGAAATACGCCTGCAACAACGAGTACTGGGAGACCTTCAAGGAGAACCCCATCAGGCTGACCGAAGAGAACGTGGTGAACTACATCACCAAGCTGAAGGGCCCCAAGGCCGCTGCCCTGTTCGCTAAGACCCACAACCTGAACATGCTGCAGGACATCCCAATGGACAGGTTCGTGATGGACCTGAAGAGGGACGTGAAGGTGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGTGCAGGTGATCCAGGCCGCTGACCCACTGGCCACCGCCTACCTGTGCGGCATCCACAGGGAGCTGGTGAGGCGGCTGAACGCCGTGCTGCTGCCCAACATCCACACCCTGTTCGACATGAGCGCCGAGGACTTCGACGCCATCATCGCCGAGCACTTCCAGCCCGGCGACTGCGTGCTGGAGACCGACATCGCCAGCTTCGACAAGAGCGAGGATGACGCTATGGCCCTGACCGCTCTGATGATCCTGGAGGACCTGGGCGTGGACGCCGAGCTGCTCACCCTGATCGAGGCTGCCTTCGGCGAGATCAGCTCCATCCACCTGCCCACCAAGACCAAGTTCAAGTTCGGCGCTATGATGAAAAGCGGAATGTTCCTGACCCTGTTCGTGAACACCGTGATCAACATTGTGATCGCCAGCAGGGTGCTGCGGGAGAGGCTGACCGGCAGCCCCTGCGCTGCCTTCATCGGCGACGACAACATCGTGAAGGGCGTGAAAAGCGACAAGCTGATGGCCGACAGGTGCGCCACCTGGCTGAACATGGAGGTGAAGATCATCGACGCCGTGGTGGGCGAGAAGGCCCCCTACTTCTGCGGCGGATTCATCCTGTGCGACAGCGTGACCGGCACCGCCTGCAGGGTGGCCGACCCCCTGAAGAGGCTGTTCAAGCTGGGCAAGCCACTGGCCGCTGACGATGAGCACGACGATGACAGGCGGAGGGCCCTGCACGAGGAAAGCACCAGGTGGAACAGGGTGGGCATCCTGAGCGAGCTGTGCAAGGCCGTGGAGAGCAGGTACGAGACCGTGGGCACCAGCATCATCGTGATGGCTATGACCACACTGGCCAGCTCCGTCAAGAGCTTCTCCTACCTG AGGGGGGCCCCTATAACTCTCTACGGCTAASTARR™ non- amino acid MEKVHVDIEEDSPFLRALQRSFPQFEVEAKQVTDNDHAN (SEQ IDstructural ARAFSHLASKLIETEVDPSDTILDIGSAPARRMYSKHKYH NO: 51) gene ORFCICPMRCAEDPDRLYKYATKLKKNCKEITDKELDKKMKELAAVMSDPDLETETMCLHDDESCRYEGQVAVYQDVYAVDGPTSLYHQANKGVRVAYWIGFDTTPFMFKNLAGAYPSYSTNWADETVLTARNIGLCSSDVMERSRRGMSILRKKYLKPSNNVLFSVGSTIYHEKRDLLRSWHLPSVFHLRGKQNYTCRCETIVSCDGYVVKRIAISPGLYGKPSGYAATMHREGFLCCKVTDTLNGERVSFPVCTYVPATLCDQMTGILATDVSADDAQKLLVGLNQRIVVNGRTQRNTNTMKNYLLPVVAQAFARWAKEYKEDQEDERPLGLRDRQLVMGCCWAFRRHKITSIYKRPDTQTIIKVNSDFHSFVLPRIGSNTLEIGLRTRIRKMLEEHKEPSPLITAEDVQEAKCAADEAKEVREAEELRAALPPLAADVEEPTLEADVDLMLQEAGAGSVETPRGLIKVTSYDGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARLFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGCAPSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVTSGATSAETNSYFAKSMEFLARPVPAPRTVFRNPPHPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITREELEALTPSRTPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQRRFDAGAYIFSSDTGQGHLQQKSVRQTVLSEVVLERTELEISYAPRLDQEKEELLRKKLQLNPTPANRSRYQSRKVENMKAITARRILQGLGHYLKAEGKVECYRTLHPVPLYSSSVNRAFSSPKVAVEACNAMLKENFPTVASYCIIPEYDAYLDMVDGASCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGPKAAALFAKTHNLNMLQDIPMDRFVMDLKRDVKVTPGTKHTEERPKVQVIQAADPLATAYLCGIHRELVRRLNAVLLPNIHTLFDMSAEDFDAIIAEHFQPGDCVLETDIASFDKSEDDAMALTALMILEDLGVDAELLTLIEAAFGEISSIHLPTKTKFKFGAMMKSGMFLTLFVNTVINIVIASRVLRERLTGSPCAAFIGDDNIVKGVKSDKLMADRCATWLNMEVKIIDAVVGEKAPYFCGGFILCDSVTGTACRVADPLKRLFKLGKPLAADDEHDDDRRRALHEESTRWNRVGILSELCKAVESRYETVGTSIIVMAMTTLASSV KSFSYLRGAPITLYG* STARR™intergenic nucleotide CCTGAATGGACTACGACATAGTCTAGTCCGCCAAGGC (SEQ IDregion CGCCACC NO: 52) STARR™ transgene nucleotiden/a (depends on gene of our interest) ORF STARR™ 3' UTR nucleotideACTCGAGTATGTTACGTGCAAAGGTGATTGTCACCCCC (SEQ IDCGAAAGACCATATTGTGACACACCCTCAGTATCACGC NO: 53)CCAAACATTTACAGCCGCGGTGTCAAAAACCGCGTGGACGTGGTTAACATCCCTGCTGGGAGGATCAGCCGTAATTATTATAATTGGCTTGGTGCTGGCTACTATTGTGGCCATGTACGTGCTGACCAACCAGAAACATAATTGAATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAATCTAGAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA Comparitive5' UTR nucleotide unknown Original non- nucleotideATGCCCGAGAAGGTGCACGTGGACATCGAGGAGGACA (SEQ ID structuralGCCCCTTCCTGAGGGCCCTGCAGAGGAGCTTCCCACA NO: 54) gene ORFGTTCGAAGTGGAGGCCAAGCAGGTGACCGACAACGACCACGCCAACGCCAGGGCCTTCAGCCACCTGGCCAGCAAGCTGATCGAGACCGAGGTGGACCCCAGCGACACCATCCTGGACATCGGCAGCGCCCCAGCCAGGAGAATGTACAGCAAGCACAAGTACCACTGCATCTGCCCCATGAGGTGCGCCGAGGACCCCGACAGGCTGTACAAGTACGCCACCAAACTGAAGAAGAACTGCAAGGAGATCACCGACAAGGAGCTGGACAAGAAAATGAAGGAGCTGGCCGCCGTGATGAGCGACCCCGACCTGGAGACCGAGACAATGTGCCTGCACGACGACGAGAGCTGCAGGTACGAGGGCCAGGTGGCCGTCTACCAGGACGTGTACGCCGTCGACGGCCCCACCAGCCTGTACCACCAGGCCAACAAGGGCGTGAGGGTGGCCTACTGGATCGGCTTCGACACCACACCCTTCATGTTCAAGAACCTGGCCGGCGCCTACCCCAGCTACAGCACCAACTGGGCCGACGAGACCGTGCTGACCGCCAGGAACATCGGCCTGTGCAGCAGCGACGTGATGGAGAGGAGCCGGAGAGGCATGAGCATCCTGAGGAAGAAATACCTGAAGCCCAGCAACAACGTGCTGTTCAGCGTGGGCAGCACCATCTACCACGAGAAGAGGGACCTGCTCAGGAGCTGGCACCTGCCCAGCGTGTTCCACCTGAGGGGCAAGCAGAACTACACCTGCAGGTGCGAGACCATCGTGAGCTGCGACGGCTACGTGGTGAAGAGGATCGCCATCAGCCCCGGCCTGTACGGCAAGCCCAGCGGCTACGCCGCTACAATGCACAGGGAGGGCTTCCTGTGCTGCAAGGTGACCGACACCCTGAACGGCGAGAGGGTGAGCTTCCCCGTGTGCACCTACGTGCCCGCCACCCTGTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGAGCGCCGACGACGCCCAGAAGCTGCTCGTGGGCCTGAACCAGAGGATCGTGGTCAACGGCAGGACCCAGAGGAACACCAACACAATGAAGAACTACCTGCTGCCCGTGGTGGCCCAGGCTTTCGCCAGGTGGGCCAAGGAGTACAAGGAGGACCAGGAAGACGAGAGGCCCCTGGGCCTGAGGGACAGGCAGCTGGTGATGGGCTGCTGCTGGGCCTTCAGGCGGCACAAGATCACCAGCATCTACAAGAGGCCCGACACCCAGACCATCATCAAGGTGAACAGCGACTTCCACAGCTTCGTGCTGCCCAGGATCGGCAGCAACACCCTGGAGATCGGCCTGAGGACCCGGATCAGGAAGATGCTGGAGGAACACAAGGAGCCCAGCCCACTGATCACCGCCGAGGACGTGCAGGAGGCCAAGTGCGCTGCCGACGAGGCCAAGGAGGTGAGGGAGGCCGAGGAACTGAGGGCCGCCCTGCCACCCCTGGCTGCCGACGTGGAGGAACCCACCCTGGAAGCCGACGTGGACCTGATGCTGCAGGAGGCCGGCGCCGGAAGCGTGGAGACACCCAGGGGCCTGATCAAGGTGACCAGCTACGACGGCGAGGACAAGATCGGCAGCTACGCCGTGCTGAGCCCACAGGCCGTGCTGAAGTCCGAGAAGCTGAGCTGCATCCACCCACTGGCCGAGCAGGTGATCGTGATCACCCACAGCGGCAGGAAGGGCAGGTACGCCGTGGAGCCCTACCACGGCAAGGTGGTCGTGCCCGAGGGCCACGCCATCCCCGTGCAGGACTTCCAGGCCCTGAGCGAGAGCGCCACCATCGTGTACAACGAGAGGGAGTTCGTGAACAGGTACCTGCACCATATCGCCACCCACGGCGGAGCCCTGAACACCGACGAGGAATACTACAAGACCGTGAAGCCCAGCGAGCACGACGGCGAGTACCTGTACGACATCGACAGGAAGCAGTGCGTGAAGAAAGAGCTGGTGACCGGCCTGGGACTGACCGGCGAGCTGGTGGACCCACCCTTCCACGAGTTCGCCTACGAGAGCCTGAGGACCAGACCCGCCGCTCCCTACCAGGTGCCCACCATCGGCGTGTACGGCGTGCCCGGCAGCGGAAAGAGCGGCATCATCAAGAGCGCCGTGACCAAGAAAGACCTGGTGGTCAGCGCCAAGAAAGAGAACTGCGCCGAGATCATCAGGGACGTGAAGAAGATGAAAGGCCTGGACGTGAACGCGCGCACCGTGGACAGCGTGCTGCTGAACGGCTGCAAGCACCCCGTGGAGACCCTGTACATCGACGAGGCCTTCGCTTGCCACGCCGGCACCCTGAGGGCCCTGATCGCCATCATCAGGCCCAAGAAAGCCGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATGATGTGCCTGAAGGTGCACTTCAACCACGAGATCTGCACCCAGGTGTTCCACAAGAGCATCAGCAGGCGGTGCACCAAGAGCGTGACCAGCGTCGTGAGCACCCTGTTCTACGACAAGAAAATGAGGACCACCAACCCCAAGGAGACCAAAATCGTGATCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCTGATCCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCAGATCGACTACAAGGGCAACGAGATCATGACCGCCGCTGCCAGCCAGGGCCTGACCAGGAAGGGCGTGTACGCCGTGAGGTACAAGGTGAACGAGAACCCACTGTACGCTCCCACCAGCGAGCACGTGAACGTGCTGCTGACCAGGACCGAGGACAGGATCGTGTGGAAGACCCTGGCCGGCGACCCCTGGATCAAGACCCTGACCGCCAAGTACCCCGGCAACTTCACCGCCACCATCGAAGAGTGGCAGGCCGAGCACGACGCCATCATGAGGCACATCCTGGAGAGGCCCGACCCCACCGACGTGTTCCAGAACAAGGCCAACGTGTGCTGGGCCAAGGCCCTGGTGCCCGTGCTGAAGACCGCCGGCATCGACATGACCACAGAGCAGTGGAACACCGTGGACTACTTCGAGACCGACAAGGCCCACAGCGCCGAGATCGTGCTGAACCAGCTGTGCGTGAGGTTCTTCGGCCTGGACCTGGACAGCGGCCTGTTCAGCGCCCCCACCGTGCCACTGAGCATCAGGAACAACCACTGGGACAACAGCCCCAGCCCAAACATGTACGGCCTGAACAAGGAGGTGGTCAGGCAGCTGAGCAGGCGGTACCCACAGCTGCCCAGGGCCGTGGCCACCGGCAGGGTGTACGACATGAACACCGGCACCCTGAGGAACTACGACCCCAGGATCAACCTGGTGCCCGTGAACAGGCGGCTGCCCCACGCCCTGGTGCTGCACCACAACGAGCACCCACAGAGCGACTTCAGCTCCTTCGTGAGCAAGCTGAAAGGCAGGACCGTGCTGGTCGTGGGCGAGAAGCTGAGCGTGCCCGGCAAGATGGTGGACTGGCTGAGCGACAGGCCCGAGGCCACCTTCCGGGCCAGGCTGGACCTCGGCATCCCCGGCGACGTGCCCAAGTACGACATCATCTTCGTGAACGTCAGGACCCCATACAAGTACCACCATTACCAGCAGTGCGAGGACCACGCCATCAAGCTGAGCATGCTGACCAAGAAGGCCTGCCTGCACCTGAACCCCGGAGGCACCTGCGTGAGCATCGGCTACGGCTACGCCGACAGGGCCAGCGAGAGCATCATTGGCGCCATCGCCAGGCTGTTCAAGTTCAGCAGGGTGTGCAAACCCAAGAGCAGCCTGGAGGAAACCGAGGTGCTGTTCGTGTTCATCGGCTACGACCGGAAGGCCAGGACCCACAACCCCTACAAGCTGAGCAGCACCCTGACAAACATCTACACCGGCAGCAGGCTGCACGAGGCCGGCTGCGCCCCCAGCTACCACGTGGTCAGGGGCGATATCGCCACCGCCACCGAGGGCGTGATCATCAACGCTGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGTGTGCGGCGCCCTGTACAAGAAGTTCCCCGAGAGCTTCGACCTGCAGCCCATCGAGGTGGGCAAGGCCAGGCTGGTGAAGGGCGCCGCTAAGCACATCATCCACGCCGTGGGCCCCAACTTCAACAAGGTGAGCGAGGTGGAAGGCGACAAGCAGCTGGCCGAAGCCTACGAGAGCATCGCCAAGATCGTGAACGACAATAACTACAAGAGCGTGGCCATCCCACTGCTCAGCACCGGCATCTTCAGCGGCAACAAGGACAGGCTGACCCAGAGCCTGAACCACCTGCTCACCGCCCTGGACACCACCGATGCCGACGTGGCCATCTACTGCAGGGACAAGAAGTGGGAGATGACCCTGAAGGAGGCCGTGGCCAGGCGGGAGGCCGTGGAAGAGATCTGCATCAGCGACGACTCCAGCGTGACCGAGCCCGACGCCGAGCTGGTGAGGGTGCACCCCAAGAGCTCCCTGGCCGGCAGGAAGGGCTACAGCACCAGCGACGGCAAGACCTTCAGCTACCTGGAGGGCACCAAGTTCCACCAGGCCGCTAAGGACATCGCCGAGATCAACGCTATGTGGCCCGTGGCCACCGAGGCCAACGAGCAGGTGTGCATGTACATCCTGGGCGAGAGCATGTCCAGCATCAGGAGCAAGTGCCCCGTGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCTGCCCTGCCTGTGCATCCACGCTATGACACCCGAGAGGGTGCAGCGGCTGAAGGCCAGCAGGCCCGAGCAGATCACCGTGTGCAGCTCCTTCCCACTGCCCAAGTACAGGATCACCGGCGTGCAGAAGATCCAGTGCAGCCAGCCCATCCTGTTCAGCCCAAAGGTGCCCGCCTACATCCACCCCAGGAAGTACCTGGTGGAGACCCCACCCGTGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCTGATCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCATCATTATCGAGGAAGAGGAAGAGGACAGCATCAGCCTGCTGAGCGACGGCCCCACCCACCAGGTGCTGCAGGTGGAGGCCGACATCCACGGCCCACCCAGCGTGTCCAGCTCCAGCTGGAGCATCCCACACGCCAGCGACTTCGACGTGGACAGCCTGAGCATCCTGGACACCCTGGAGGGCGCCAGCGTGACCTCCGGCGCCACCAGCGCCGAGACCAACAGCTACTTCGCCAAGAGCATGGAGTTCCTGGCCAGGCCCGTGCCAGCTCCCAGGACCGTGTTCAGGAACCCACCCCACCCAGCTCCCAGGACCAGGACCCCAAGCCTGGCTCCCAGCAGGGCCTGCAGCAGGACCAGCCTGGTGAGCACCCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAACTGGAGGCCCTGACACCCAGCAGGACCCCCAGCAGGTCCGTGAGCAGGACTAGTCTGGTGTCCAACCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAATTCGAGGCCTTCGTGGCCCAGCAACAGAGACGGTTCGACGCCGGCGCCTACATCTTCAGCAGCGACACCGGCCAGGGACACCTGCAGCAAAAGAGCGTGAGGCAGACCGTGCTGAGCGAGGTGGTGCTGGAGAGGACCGAGCTGGAAATCAGCTACGCCCCCAGGCTGGACCAGGAGAAGGAGGAACTGCTCAGGAAGAAACTGCAGCTGAACCCCACCCCAGCCAACAGGAGCAGGTACCAGAGCAGGAAGGTGGAGAACATGAAGGCCATCACCGCCAGGCGGATCCTGCAGGGCCTGGGACACTACCTGAAGGCCGAGGGCAAGGTGGAGTGCTACAGGACCCTGCACCCCGTGCCACTGTACAGCTCCAGCGTGAACAGGGCCTTCTCCAGCCCCAAGGTGGCCGTGGAGGCCTGCAACGCTATGCTGAAGGAGAACTTCCCCACCGTGGCCAGCTACTGCATCATCCCCGAGTACGACGCCTACCTGGACATGGTGGACGGCGCCAGCTGCTGCCTGGACACCGCCAGCTTCTGCCCCGCCAAGCTGAGGAGCTTCCCCAAGAAACACAGCTACCTGGAGCCCACCATCAGGAGCGCCGTGCCCAGCGCCATCCAGAACACCCTGCAGAACGTGCTGGCCGCTGCCACCAAGAGGAACTGCAACGTGACCCAGATGAGGGAGCTGCCCGTGCTGGACAGCGCTGCCTTCAACGTGGAGTGCTTCAAGAAATACGCCTGCAACAACGAGTACTGGGAGACCTTCAAGGAGAACCCCATCAGGCTGACCGAAGAGAACGTGGTGAACTACATCACCAAGCTGAAGGGCCCCAAGGCCGCTGCCCTGTTCGCTAAGACCCACAACCTGAACATGCTGCAGGACATCCCAATGGACAGGTTCGTGATGGACCTGAAGAGGGACGTGAAGGTGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGTGCAGGTGATCCAGGCCGCTGACCCACTGGCCACCGCCTACCTGTGCGGCATCCACAGGGAGCTGGTGAGGCGGCTGAACGCCGTGCTGCTGCCCAACATCCACACCCTGTTCGACATGAGCGCCGAGGACTTCGACGCCATCATCGCCGAGCACTTCCAGCCCGGCGACTGCGTGCTGGAGACCGACATCGCCAGCTTCGACAAGAGCGAGGATGACGCTATGGCCCTGACCGCTCTGATGATCCTGGAGGACCTGGGCGTGGACGCCGAGCTGCTCACCCTGATCGAGGCTGCCTTCGGCGAGATCAGCTCCATCCACCTGCCCACCAAGACCAAGTTCAAGTTCGGCGCTATGATGAAAAGCGGAATGTTCCTGACCCTGTTCGTGAACACCGTGATCAACATTGTGATCGCCAGCAGGGTGCTGCGGGAGAGGCTGACCGGCAGCCCCTGCGCTGCCTTCATCGGCGACGACAACATCGTGAAGGGCGTGAAAAGCGACAAGCTGATGGCCGACAGGTGCGCCACCTGGCTGAACATGGAGGTGAAGATCATCGACGCCGTGGTGGGCGAGAAGGCCCCCTACTTCTGCGGCGGATTCATCCTGTGCGACAGCGTGACCGGCACCGCCTGCAGGGTGGCCGACCCCCTGAAGAGGCTGTTCAAGCTGGGCAAGCCACTGGCCGCTGACGATGAGCACGACGATGACAGGCGGAGGGCCCTGCACGAGGAAAGCACCAGGTGGAACAGGGTGGGCATCCTGAGCGAGCTGTGCAAGGCCGTGGAGAGCAGGTACGAGACCGTGGGCACCAGCATCATCGTGATGGCTATGACCACACTGGCCAGCTCCGTCAAGAGCTTCTCCTACCTGAGGGGGGCCCCTATAACTCTCTACGGCT AA Comparitive non- amino acidMPEKVHVDIEEDSPFLRALQRSFPQFEVEAKQVTDNDHA (SEQ ID structuralNARAFSHLASKLIETEVDPSDTILDIGSAPARRMYSKHKY NO: 55) gene ORFHCICPMRCAEDPDRLYKYATKLKKNCKEITDKELDKKMKELAAVMSDPDLETETMCLHDDESCRYEGQVAVYQDVYAVDGPTSLYHQANKGVRVAYWIGFDTTPFMFKNLAGAYPSYSTNWADETVLTARNIGLCSSDVMERSRRGMSILRKKYLKPSNNVLFSVGSTIYHEKRDLLRSWHLPSVFHLRGKQNYTCRCETIVSCDGYVVKRIAISPGLYGKPSGYAATMHREGFLCCKVTDTLNGERVSFPVCTYVPATLCDQMTGILATDVSADDAQKLLVGLNQRIVVNGRTQRNTNTMKNYLLPVVAQAFARWAKEYKEDQEDERPLGLRDRQLVMGCCWAFRRHKITSIYKRPDTQTIIKVNSDFHSFVLPRIGSNTLEIGLRTRIRKMLEEHKEPSPLITAEDVQEAKCAADEAKEVREAEELRAALPPLAADVEEPTLEADVDLMLQEAGAGSVETPRGLIKVTSYDGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARLFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGCAPSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPINIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVTSGATSAETNSYFAKSMEFLARPVPAPRTVFRNPPHPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITREELEALTPSRTPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQRRFDAGAYIFSSDTGQGHLQQKSVRQTVLSEVVLERTELEISYAPRLDQEKEELLRKKLQLNPTPANRSRYQSRKVENMKAITARRILQGLGHYLKAEGKVECYRTLHPVPLYSSSVNRAFSSPKVAVEACNAMLKENFPTVASYCIIPEYDAYLDMVDGASCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGPKAAALFAKTHNLNMLQDIPMDRFVMDLKRDVKVTPGTKHTEERPKVQVIQAADPLATAYLCGIHRELVRRLNAVLLPNIHTLFDMSAEDFDAIIAEHFQPGDCVLETDIASFDKSEDDAMALTALMILEDLGVDAELLTLIEAAFGEISSIHLPTKTKFKFGAMMKSGMFLTLFVNTVINIVIASRVLRERLTGSPCAAFIGDDNIVKGVKSDKLMADRCATWLNMEVKIIDAVVGEKAPYFCGGFILCDSVTGTACRVADPLKRLFKLGKPLAADDEHDDDRRRALHEESTRWNRVGILSELCKAVESRYETVGTSIIVMAMTTL ASSVKSFSYLRGAPITLYG*Comparitive intergenic nucleotide unknown region Comparitive 3' UTRnucleotide unknown

TABLE 5 ORF of Peptide of Interest forSelf-Replicating RNAs of the Disclosure ORF Sequence Identity TypeSequence 2019-nCoV nucleotideATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGT Spike geneTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAATTC (SEQ IDTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCA NO: 117)GTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTITTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCA AATTACATTACACATAA 2019-nCoVamino acid MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSV Spike geneLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFAS (SEQ IDTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG NO: 118)VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQUITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT* 2019-nCoV nucleotideATGTTCGTCTTCCTGGTCCTGCTGCCTCTGGTCTCCTCACAGTGCG Spike geneTCAATCTGACAACTCGGACTCAGCTGCCACCTGCTTATACTAATA (SEQ IDGCTTCACCAGAGGCGTGTACTATCCTGACAAGGTGTTTAGAAGCT NO: 119)CCGTGCTGCACTCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAATGGCACAAAGCGGTTCGACAATCCCGTGCTGCCTTTTAACGATGGCGTGTACTTCGCCTCTACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTTGGCACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGTCATCAAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAATAAGAGCTGGATGGAGTCCGAGTTTAGAGTGTATTCTAGCGCCAACAACTGCACATTTGAGTACGTGAGCCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGACGGCTACTTCAAAATCTACTCTAAGCACACCCCCATCAACCTGGTGCGCGACCTGCCTCAGGGCTTCAGCGCCCTGGAGCCCCTGGTGGATCTGCCTATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAGCGGATGGACCGCCGGCGCTGCCGCCTACTATGTGGGCTACCTCCAGCCCCGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATCACAGACGCAGTGGATTGCGCCCTGGACCCCCTGAGCGAGACAAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATCAGACATCCAATTTCAGGGTGCAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGTTCAACGCAACCCGCTTCGCCAGCGTGTACGCCTGGAATAGGAAGCGGATCAGCAACTGCGTGGCCGACTATAGCGTGCTGTACAACTCCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACCTGTGCTTTACCAACGTCTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGCCCCCGGCCAGACAGGCAAGATCGCAGACTACAATTATAAGCTGCCAGACGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGGCAACTACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCTACAGAAATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCCACTCCAGTCCTACGGCTTCCAGCCCACAAACGGCGTGGGCTATCAGCCTTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCCCCAGCAACAGTGTGCGGCCCCAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGCACAGGCGTGCTGACCGAGTCCAACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGACCCACAGACCCTGGAGATCCTGGACATCACACCCTGCTCTTTCGGCGGCGTGAGCGTGATCACACCCGGCACCAATACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGTACCGAGGTGCCCGTGGCTATCCACGCCGATCAGCTGACCCCAACATGGCGGGTGTACAGCACCGGCTCCAACGTCTTCCAGACAAGAGCCGGATGCCTGATCGGAGCAGAGCACGTGAACAATTCCTATGAGTGCGACATCCCAATCGGCGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCCAGAAGAGCCCGGAGCGTGGCCTCCCAGTCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAACAGCGTGGCCTACTCTAACAATAGCATCGCCATCCCAACCAACTTCACAATCTCTGTGACCACAGAGATCCTGCCCGTGTCCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTACCGAGTGCAGCAACCTGCTGCTCCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCCCTGACAGGCATCGCCGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAAATCTACAAGACCCCCCCTATCAAGGACTTTGGCGGCTTCAATTTTTCCCAGATCCTGCCTGATCCATCCAAGCCTTCTAAGCGGAGCTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCAGCCAGGGACCTGATCTGCGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCACCCCTGCTGACAGATGAGATGATCGCACAGTACACAAGCGCCCTGCTGGCCGGCACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTCCAGATCCCCTTTGCCATGCAGATGGCCTATAGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCTGTCCTCTACAGCCAGCGCCCTGGGCAAGCTCCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCATCTCTAGCGTGCTGAATGACATCCTGAGCCGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACCGGCCGGCTCCAGAGCCTCCAGACCTATGTGACACAGCAGCTGATCAGGGCCGCCGAGATCAGGGCCAGCGCCAATCTGGCAGCAACCAAGATGTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGGGCTATCACCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTGGTGTTTCTGCACGTGACCTACGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAAGGCCCACTTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCTACGAGCCCCAGATCATCACCACAGACAACACCTTCGTGAGCGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCACTCCAGCCCGAGCTGGACAGCTTTAAGGAGGAGCTGGATAAGTATTTCAAGAATCACACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCTAAGAATCTGAACGAGAGCCTGATCGACCTCCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGACATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGACGAGGATGACTCTGAACCTGTGCTGAAGGGCGTGAAG CTGCATTACACCTAA 2019-nCoVamino acid MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSV Spike geneLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFAS (SEQ IDTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG NO: 120)VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQUITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT*

RNA sequences can include any combination of the RNA sequences listed inTables 4 and 5. In some embodiments, RNA sequences of the presentdisclosure include any combination of the RNA sequences listed in Tables4 and 5 in which 0% to 100%, 1% to 100%, 25% to 100%, 50% to 100% and75% to 100% of the uracil nucleotides of the mRNA sequences aremodified. In some embodiments, 1% to 100% of the uracil nucleotides areN1-methylpseudouridine or 5-methoxyuridine. In some embodiments, 100/%of the uracil nucleotides are N1-methylpseudouridine. In someembodiments, 100% of the uracil nucleotides are 5-methoxyuridine.

A self-replicating RNA of the disclosure may be obtained by any suitablemeans. Methods for the manufacture of self-replicating RNA are known inthe art and would be readily apparent to a person of ordinary skill. Aself-replicating RNA of the disclosure may be prepared according to anyavailable technique including, but not limited to chemical synthesis, invitro transcription (IVT) or enzymatic or chemical cleavage of a longerprecursor, etc.

In some embodiments, a self-replicating RNA of the disclosure isproduced from a primary complementary DNA (cDNA) construct. The cDNAconstructs can be produced on an RNA template by the action of a reversetranscriptase (e.g., RNA-dependent DNA-polymerase). The process ofdesign and synthesis of the primary cDNA constructs described hereingenerally includes the steps of gene construction, RNA production(either with or without modifications) and purification. In the IVTmethod, a target polynucleotide sequence encoding a self-replicating RNAof the disclosure is first selected for incorporation into a vectorwhich will be amplified to produce a cDNA template. Optionally, thetarget polynucleotide sequence and/or any flanking sequences may becodon optimized. The cDNA template is then used to produce aself-replicating RNA of the disclosure through in vitro transcription(IVT). After production, the self-replicating RNA of the disclosure mayundergo purification and clean-up processes. The steps of which areprovided in more detail below.

The step of gene construction may include, but is not limited to genesynthesis, vector amplification, plasmid purification, plasmidlinearization and clean-up, and cDNA template synthesis and clean-up.Once a protein of interest is selected for production, a primaryconstruct is designed. Within the primary construct, a first region oflinked nucleosides encoding the polypeptide of interest may beconstructed using an open reading frame (ORF) of a selected nucleic acid(DNA or RNA) transcript. The ORF may comprise the wild type ORF, anisoform, variant or a fragment thereof. As used herein, an “open readingframe” or “ORF” is meant to refer to a nucleic acid sequence (DNA orRNA) which is capable of encoding a polypeptide of interest. ORFs oftenbegin with the start codon, ATG and end with a nonsense or terminationcodon or signal.

The cDNA templates may be transcribed to produce a self-replicating RNAof the disclosure using an in vitro transcription (IVT) system. Thesystem typically comprises a transcription buffer, nucleotidetriphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs maybe selected from, but are not limited to, those described hereinincluding natural and unnatural (modified) NTPs. The polymerase may beselected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to incorporate modified nucleic acids.

The primary cDNA template or transcribed RNA sequence may also undergocapping and/or tailing reactions. A capping reaction may be performed bymethods known in the art to add a 5′ cap to the 5′ end of the primaryconstruct. Methods for capping include, but are not limited to, using aVaccinia Capping enzyme (New England Biolabs, Ipswich, Mass.) or cappingat initiation of in vitro transcription, by for example, including acapping agent as part of the IVT reaction. (Nuc. Acids Symp. (2009)53:129). A poly(A) tailing reaction may be performed by methods known inthe art, such as, but not limited to, 2′ O-methyltransferase and bymethods as described herein. If the primary construct generated fromcDNA does not include a poly-T, it may be beneficial to perform thepoly(A)-tailing reaction before the primary construct is cleaned.

Codon optimized cDNA constructs encoding the non-structural proteins andthe transgene for a self-replicating RNA protein are particularlysuitable for generating self-replicating RNA sequences described herein.For example, such cDNA constructs may be used as the basis totranscribe, in vitro, a polyribonucleotide encoding a protein ofinterest as part of a self-replicating RNA.

The present disclosure also provides expression vectors comprising anucleotide sequence encoding a self-replicating RNA that is preferablyoperably linked to at least one regulatory sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe encoded polypeptide.

Accordingly, the term regulatory sequence includes promoters, enhancers,and other expression control elements. The design of the expressionvector may depend on such factors as the choice of the host cell to betransformed and/or the type of protein desired to be expressed.

The present disclosure also provides polynucleotides (e.g. DNA, RNA,cDNA, mRNA, etc.) directed to a self-replicating RNA of the disclosurethat may be operably linked to one or more regulatory nucleotidesequences in an expression construct, such as a vector or plasmid. Incertain embodiments, such constructs are DNA constructs. Regulatorynucleotide sequences will generally be appropriate for a host cell usedfor expression. Numerous types of appropriate expression vectors andsuitable regulatory sequences are known in the art for a variety of hostcells.

Typically, said one or more regulatory nucleotide sequences may include,but are not limited to, promoter sequences, leader or signal sequences,ribosomal binding sites, transcriptional start and terminationsequences, translational start and termination sequences, and enhanceror activator sequences. Constitutive or inducible promoters as known inthe art are contemplated by the embodiments of the present disclosure.The promoters may be either naturally occurring promoters, or hybridpromoters that combine elements of more than one promoter.

An expression construct may be present in a cell on an episome, such asa plasmid, or the expression construct may be inserted in a chromosome.In some embodiments, the expression vector contains a selectable markergene to allow the selection of transformed host cells. Selectable markergenes are well known in the art and will vary with the host cell used.

The present disclosure also provides a host cell transfected with aself-replicating RNA or DNA described herein. The self-replicating RNAor DNA can encode any coronavirus protein of interest, for example anantigen, including the S-antigen of the COVID-19 virus. The host cellmay be any prokaryotic or eukaryotic cell. For example, a polypeptideencoded by a self-replicating RNA may be expressed in bacterial cellssuch as E. coli, insect cells (e.g., using a baculovirus expressionsystem), yeast, or mammalian cells. Other suitable host cells are knownto those skilled in the art.

A host cell transfected with an expression vector comprising aself-replicating RNA of the disclosure can be cultured under appropriateconditions to allow expression of the amplification of theself-replicating RNA and translation of the polypeptide to occur. Thepolypeptide may be secreted and isolated from a mixture of cells andmedium containing the polypeptides. Alternatively, the polypeptides maybe retained in the cytoplasm or in a membrane fraction and the cellsharvested, lysed and the protein isolated. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art.

The expressed proteins described herein can be isolated from cellculture medium, host cells, or both using techniques known in the artfor purifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the polypeptide.

Compostions and Pharmaceutical Compostions

Provided herein, in some embodiments, are compositions comprising any ofthe nucleic acid molecules provided herein. Compositions provided hereincan include a lipid. Any lipid can be included in compositions providedherein. In one aspect, the lipid is an ionizable cationic lipid. Anyionizable cationic lipid can be included in compositions comprisingnucleic acid molecules provided herein.

The compositions and polynucleotides of the present disclosure may beused to immunize or vaccinate a subject against a viral infection. Insome embodiments, the compositions and polynucleotides of the presentdisclosure may be used to vaccinate or immunize a subject againstCOVID-19 virus.

Also provided herein, in some embodiments, are pharmaceuticalcompositions comprising any of the nucleic acid molecules providedherein and a lipid formulation. Any lipid can be included in lipidformulations of pharmaceutical compositions provided herein. In oneaspect, lipid formulations of pharmaceutical compositions providedherein include an ionizable cationic lipid. Exemplary ionizable cationiclipids of compositions and pharmaceutical compositions provided hereininclude the following:

In one aspect, the ionizable cationic lipid of compositions providedherein has a structure of

or a pharmaceutically acceptable salt thereof.

In another aspect, the ionizable cationic lipid of compositions providedherein has a structure of

or a pharmaceutically acceptable salt thereof.

In one aspect, the ionizable cationic lipid included in lipidformulations of pharmaceutical compositions provided herein has astructure of

or a pharmaceutically acceptable salt thereof.

In another aspect, the ionizable cationic lipid included in lipidformulations of pharmaceutical compositions provided herein has astructure of

or a pharmaceutically acceptable salt thereof.

Lipid Formulations/LNPs

Therapies based on the intracellular delivery of nucleic acids to targetcells face both extracellular and intracellular barriers. Indeed, nakednucleic acid materials cannot be easily systemically administered due totheir toxicity, low stability in serum, rapid renal clearance, reduceduptake by target cells, phagocyte uptake and their ability in activatingthe immune response, all features that preclude their clinicaldevelopment. When exogenous nucleic acid material (e.g., mRNA) entersthe human biological system, it is recognized by the reticuloendothelialsystem (RES) as foreign pathogens and cleared from blood circulationbefore having the chance to encounter target cells within or outside thevascular system. It has been reported that the half-life of nakednucleic acid in the blood stream is around several minutes (Kawabata K,Takakura Y, Hashida MPharm Res. 1995 June; 12(6):825-30). Chemicalmodification and a proper delivery method can reduce uptake by the RESand protect nucleic acids from degradation by ubiquitous nucleases,which increase stability and efficacy of nucleic acid-based therapies.In addition, RNAs or DNAs are anionic hydrophilic polymers that are notfavorable for uptake by cells, which are also anionic at the surface.The success of nucleic acid-based therapies thus depends largely on thedevelopment of vehicles or vectors that can efficiently and effectivelydeliver genetic material to target cells and obtain sufficient levels ofexpression in vivo with minimal toxicity.

Moreover, upon internalization into a target cell, nucleic acid deliveryvectors are challenged by intracellular barriers, including endosomeentrapment, lysosomal degradation, nucleic acid unpacking from vectors,translocation across the nuclear membrane (for DNA), release at thecytoplasm (for RNA), and so on. Successful nucleic acid-based therapythus depends upon the ability of the vector to deliver the nucleic acidsto the target sites inside of the cells in order to obtain sufficientlevels of a desired activity such as expression of a gene.

While several gene therapies have been able to successfully utilize aviral delivery vector (e.g., AAV), lipid-based formulations have beenincreasingly recognized as one of the most promising delivery systemsfor RNA and other nucleic acid compounds due to their biocompatibilityand their ease of large-scale production. One of the most significantadvances in lipid-based nucleic acid therapies happened in August 2018when Patisiran (ALN-TTR02) was the first siRNA therapeutic approved bythe Food and Drug Administration (FDA) and by the European Commission(EC). ALN-TTR02 is an siRNA formulation based upon the so-called StableNucleic Acid Lipid Particle (SNALP) transfecting technology. Despite thesuccess of Patisiran, the delivery of nucleic acid therapeutics,including mRNA, via lipid formulations is still under ongoingdevelopment.

Some art-recognized lipid-formulated delivery vehicles for nucleic acidtherapeutics include, according to various embodiments, polymer basedcarriers, such as polyethyleneimine (PEI), lipid nanoparticles andliposomes, nanoliposomes, ceramide-containing nanoliposomes,multivesicular liposomes, proteoliposomes, both natural andsynthetically-derived exosomes, natural, synthetic and semi-syntheticlamellar bodies, nanoparticulates, micelles, and emulsions. These lipidformulations can vary in their structure and composition, and as can beexpected in a rapidly evolving field, several different terms have beenused in the art to describe a single type of delivery vehicle. At thesame time, the terms for lipid formulations have varied as to theirintended meaning throughout the scientific literature, and thisinconsistent use has caused confusion as to the exact meaning of severalterms for lipid formulations. Among the several potential lipidformulations, liposomes, cationic liposomes, and lipid nanoparticles arespecifically described in detail and defined herein for the purposes ofthe present disclosure.

Liposomes

Conventional liposomes are vesicles that consist of at least one bilayerand an internal aqueous compartment. Bilayer membranes of liposomes aretypically formed by amphiphilic molecules, such as lipids of syntheticor natural origin that comprise spatially separated hydrophilic andhydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998).Bilayer membranes of the liposomes can also be formed by amphiphilicpolymers and surfactants (e.g., polymerosomes, niosomes, etc.). Theygenerally present as spherical vesicles and can range in size from 20 nmto a few microns. Liposomal formulations can be prepared as a colloidaldispersion or they can be lyophilized to reduce stability risks and toimprove the shelf-life for liposome-based drugs. Methods of preparingliposomal compositions are known in the art and would be within theskill of an ordinary artisan.

Liposomes that have only one bilayer are referred to as beingunilamellar, and those having more than one bilayer are referred to asmultilamellar. The most common types of liposomes are small unilamellarvesicles (SUV), large unilamellar vesicle (LUV), and multilamellarvesicles (MLV). In contrast to liposomes, lysosomes, micelles, andreversed micelles are composed of monolayers of lipids. Generally, aliposome is thought of as having a single interior compartment, howeversome formulations can be multivesicular liposomes (MVL), which consistof numerous discontinuous internal aqueous compartments separated byseveral nonconcentric lipid bilayers.

Liposomes have long been perceived as drug delivery vehicles because oftheir superior biocompatibility, given that liposomes are basicallyanalogs of biological membranes, and can be prepared from both naturaland synthetic phospholipids (Int J Nanomedicine. 2014; 9:1833-1843). Intheir use as drug delivery vehicles, because a liposome has an aqueoussolution core surrounded by a hydrophobic membrane, hydrophilic solutesdissolved in the core cannot readily pass through the bilayer, andhydrophobic compounds will associate with the bilayer. Thus, a liposomecan be loaded with hydrophobic and/or hydrophilic molecules. When aliposome is used to carry a nucleic acid such as RNA, the nucleic acidwill be contained within the liposomal compartment in an aqueous phase.

Cationic Liposomes

Liposomes can be composed of cationic, anionic, and/or neutral lipids.As an important subclass of liposomes, cationic liposomes are liposomesthat are made in whole or part from positively charged lipids, or morespecifically a lipid that comprises both a cationic group and alipophilic portion. In addition to the general characteristics profiledabove for liposomes, the positively charged moieties of cationic lipidsused in cationic liposomes provide several advantages and some uniquestructural features. For example, the lipophilic portion of the cationiclipid is hydrophobic and thus will direct itself away from the aqueousinterior of the liposome and associate with other nonpolar andhydrophobic species. Conversely, the cationic moiety will associate withaqueous media and more importantly with polar molecules and species withwhich it can complex in the aqueous interior of the cationic liposome.For these reasons, cationic liposomes are increasingly being researchedfor use in gene therapy due to their favorability towards negativelycharged nucleic acids via electrostatic interactions, resulting incomplexes that offer biocompatibility, low toxicity, and the possibilityof the large-scale production required for in vivo clinicalapplications. Cationic lipids suitable for use in cationic liposomes arelisted herein below.

Lipid Nanoparticles

In contrast to liposomes and cationic liposomes, lipid nanoparticles(LNP) have a structure that includes a single monolayer or bilayer oflipids that encapsulates a compound in a solid phase. Thus, unlikeliposomes, lipid nanoparticles do not have an aqueous phase or otherliquid phase in its interior, but rather the lipids from the bilayer ormonolayer shell are directly complexed to the internal compound therebyencapsulating it in a solid core. Lipid nanoparticles are typicallyspherical vesicles having a relatively uniform dispersion of shape andsize. While sources vary on what size qualifies a lipid particle asbeing a nanoparticle, there is some overlap in agreement that a lipidnanoparticle can have a diameter in the range of from 10 nm to 1000 nm.However, more commonly they are considered to be smaller than 120 nm oreven 100 nm.

For lipid nanoparticle nucleic acid delivery systems, the lipid shell isformulated to include an ionizable cationic lipid which can complex toand associate with the negatively charged backbone of the nucleic acidcore. Ionizable cationic lipids with apparent pKa values below about 7have the benefit of providing a cationic lipid for complexing with thenucleic acid's negatively charged backbone and loading into the lipidnanoparticle at pH values below the pKa of the ionizable lipid where itis positively charged. Then, at physiological pH values, the lipidnanoparticle can adopt a relatively neutral exterior allowing for asignificant increase in the circulation half-lives of the particlesfollowing i.v. administration. In the context of nucleic acid delivery,lipid nanoparticles offer many advantages over other lipid-based nucleicacid delivery systems including high nucleic acid encapsulationefficiency, potent transfection, improved penetration into tissues todeliver therapeutics, and low levels of cytotoxicity and immunogenicity.

Prior to the development of lipid nanoparticle delivery systems fornucleic acids, cationic lipids were widely studied as syntheticmaterials for delivery of nucleic acid medicines. In these earlyefforts, after mixing together at physiological pH, nucleic acids werecondensed by cationic lipids to form lipid-nucleic acid complexes knownas lipoplexes. However, lipoplexes proved to be unstable andcharacterized by broad size distributions ranging from the submicronscale to a few microns. Lipoplexes, such as the Lipofectamine® reagent,have found considerable utility for in vitro transfection. However,these first-generation lipoplexes have not proven useful in vivo. Thelarge particle size and positive charge (Imparted by the cationic lipid)result in rapid plasma clearance, hemolytic and other toxicities, aswell as immune system activation. In some aspects, nucleic acidmolecules provided herein and lipids or lipid formulations providedherein form a lipid nanoparticle (LNP).

In other aspects, nucleic acid molecules provided herein areincorporated into a lipid formulation (i.e., a lipid-based deliveryvehicle).

In the context of the present disclosure, a lipid-based delivery vehicletypically serves to transport a desired RNA to a target cell or tissue.The lipid-based delivery vehicle can be any suitable lipid-baseddelivery vehicle known in the art. In some aspects, the lipid-baseddelivery vehicle is a liposome, a cationic liposome, or a lipidnanoparticle containing a self-replicating RNA of the disclosure. Insome aspects, the lipid-based delivery vehicle comprises a nanoparticleor a bilayer of lipid molecules and a self-replicating RNA of thedisclosure. In some aspects, the lipid bilayer further comprises aneutral lipid or a polymer. In some aspects, the lipid formulationcomprises a liquid medium. In some aspects, the formulation furtherencapsulates a nucleic acid. In some aspects, the lipid formulationfurther comprises a nucleic acid and a neutral lipid or a polymer. Insome aspects, the lipid formulation encapsulates the nucleic acid.

The description provides lipid formulations comprising one or moreself-replicating RNA molecules encapsulated within the lipidformulation. In some aspects, the lipid formulation comprises liposomes.In some aspects, the lipid formulation comprises cationic liposomes. Insome aspects, the lipid formulation comprises lipid nanoparticles.

In some aspects, the self-replicating RNA is fully encapsulated withinthe lipid portion of the lipid formulation such that the RNA in thelipid formulation is resistant in aqueous solution to nucleasedegradation. In other aspects, the lipid formulations described hereinare substantially non-toxic to animals such as humans and other mammals.

The lipid formulations of the disclosure also typically have a totallipid:RNA ratio (mass/mass ratio) of from about 1:1 to about 100:1, fromabout 1:1 to about 50:1, from about 2:1 to about 45:1, from about 3:1 toabout 40:1, from about 5:1 to about 45:1, or from about 10:1 to about40:1, or from about 15:1 to about 40:1, or from about 20:1 to about40:1; or from about 25:1 to about 45:1; or from about 30:1 to about45:1; or from about 32:1 to about 42:1; or from about 34:1 to about42:1. In some aspects, the total lipid:RNA ratio (mass/mass ratio) isfrom about 30:1 to about 45:1. The ratio may be any value or subvaluewithin the recited ranges, including endpoints.

The lipid formulations of the present disclosure typically have a meandiameter of from about 30 nm to about 150 nm, from about 40 nm to about150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm,from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm,about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about150 nm, and are substantially non-toxic. The diameter may be any valueor subvalue within the recited ranges, including endpoints. In addition,nucleic acids, when present in the lipid nanoparticles of the presentdisclosure, generally are resistant in aqueous solution to degradationwith a nuclease.

In some embodiments, the lipid nanoparticle has a size of less thanabout 500 nm, less than about 400 nm, less than about 300 nm, less thanabout 200 nm, less than about 100 nm, or less than about 50 nm. Inspecific embodiments, the lipid nanoparticle has a size of about 55 nmto about 90 nm.

In some aspects, the lipid formulations comprise a self-replicating RNA,a cationic lipid (e.g., one or more cationic lipids or salts thereofdescribed herein), a phospholipid, and a conjugated lipid that inhibitsaggregation of the particles (e.g., one or more PEG-lipid conjugates).The lipid formulations can also include cholesterol. In one aspect, thecationic lipid is an ionizable cationic lipid.

In the nucleic acid-lipid formulations, the RNA may be fullyencapsulated within the lipid portion of the formulation, therebyprotecting the nucleic acid from nuclease degradation. In some aspects,a lipid formulation comprising an RNA is fully encapsulated within thelipid portion of the lipid formulation, thereby protecting the nucleicacid from nuclease degradation. In certain aspects, the RNA in the lipidformulation is not substantially degraded after exposure of the particleto a nuclease at 37° C. for at least 20, 30, 45, or 60 minutes. Incertain other aspects, the RNA in the lipid formulation is notsubstantially degraded after incubation of the formulation in serum at37° C. for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.In some aspects, the RNA is complexed with the lipid portion of theformulation. One of the benefits of the formulations of the presentdisclosure is that the nucleic acid-lipid compositions are substantiallynon-toxic to animals such as humans and other mammals.

In the context of nucleic acids, full encapsulation may be determined byperforming a membrane-impermeable fluorescent dye exclusion assay, whichuses a dye that has enhanced fluorescence when associated with nucleicacid. Encapsulation is determined by adding the dye to a lipidformulation, measuring the resulting fluorescence, and comparing it tothe fluorescence observed upon addition of a small amount of nonionicdetergent. Detergent-mediated disruption of the lipid layer releases theencapsulated nucleic acid, allowing it to interact with themembrane-impermeable dye. Nucleic acid encapsulation may be calculatedas E=(10−1)/10, where/and 10 refers to the fluorescence intensitiesbefore and after the addition of detergent.

In some aspects, the present disclosure provides a nucleic acid-lipidcomposition comprising a plurality of nucleic acid-liposomes, nucleicacid-cationic liposomes, or nucleic acid-lipid nanoparticles. In someaspects, the nucleic acid-lipid composition comprises a plurality ofRNA-liposomes. In some aspects, the nucleic acid-lipid compositioncomprises a plurality of RNA-cationic liposomes. In some aspects, thenucleic acid-lipid composition comprises a plurality of RNA-lipidnanoparticles.

In some aspects, the lipid formulations comprise RNA that is fullyencapsulated within the lipid portion of the formulation, such that fromabout 30% to about 100%, from about 40% to about 100%, from about 50% toabout 100%, from about 60% to about 100%, from about 70% to about 100%,from about 80% to about 100%, from about 90% to about 100%, from about30% to about 95%, from about 40% to about 95%, from about 50% to about95%, from about 60% to about 95%, from about 70% to about 95%, fromabout 80% to about 95%, from about 85% to about 95%, from about 90% h toabout 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,or about 99% (or any fraction thereof or range therein) of the particleshave the RNA encapsulated therein. The amount may be any value orsubvalue within the recited ranges, including endpoints. The RNAincluded in any RNA-lipid composition or RNA-lipid formulation providedherein can be a self-replicating RNA.

Depending on the intended use of the lipid formulation, the proportionsof the components can be varied, and the delivery efficiency of aparticular formulation can be measured using assays known in the art.

In some aspects, nucleic acid molecules provided herein are lipidformulated. The lipid formulation is preferably selected from, but notlimited to, liposomes, cationic liposomes, and lipid nanoparticles. Inone aspect, a lipid formulation is a cationic liposome or a lipidnanoparticle (LNP) comprising:

-   -   (a) an RNA of the present disclosure,    -   (b) a cationic lipid,    -   (c) an aggregation reducing agent (such as polyethylene glycol        (PEG) lipid or PEG-modified lipid),    -   (d) optionally a non-cationic lipid (such as a neutral lipid),        and    -   (e) optionally, a sterol.

In another aspect, the cationic lipid is an ionizable cationic lipid.Any ionizable cationic lipid can be included in lipid formulations,including exemplary cationic lipids provided herein.

Cationic Lipids

In one aspect, the lipid nanoparticle formulation comprises (i) at leastone cationic lipid; (ii) a helper lipid; (iii) a sterol (e.g.,cholesterol); and (iv) a PEG-lipid. In another aspect, the cationiclipid is an ionizable cationic lipid. In yet another aspect, the lipidnanoparticle formulation comprises (i) at least one cationic lipid; (ii)a helper lipid; (iii) a sterol (e.g., cholesterol); and (iv) aPEG-lipid, in a molar ratio of about 40-70% ionizable cationiclipid:about 2-15% helper lipid:about 20-45% sterol; about 0.5-5%PEG-lipid. In a further aspect, the cationic lipid is an ionizablecationic lipid.

In one aspect, the lipid nanoparticle formulation consists of (i) atleast one cationic lipid; (ii) a helper lipid; (iii) a sterol (e.g.,cholesterol); and (iv) a PEG-lipid. In another aspect, the cationiclipid is an ionizable cationic lipid. In yet another aspect, the lipidnanoparticle formulation consists of (i) at least one cationic lipid;(ii) a helper lipid; (iii) a sterol (e.g., cholesterol); and (iv) aPEG-lipid, in a molar ratio of about 40-70% ionizable cationiclipid:about 2-15% helper lipid:about 20-45% sterol; about 0.5-5%PEG-lipid. In a further aspect, the cationic lipid is an ionizablecationic lipid.

In the presently disclosed lipid formulations, the cationic lipid maybe, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known asN-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and1,2-Dioleyloxy-3-trimethylaminopropane chloride salt),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),I-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanediol (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate(MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-K-C₂-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28 31-tetraen-19-yl4-(dimethylamino) butanoate (DLin-M-C3-DMA),3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine(MC3 Ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine(MC4 Ether), or any combination thereof. Other cationic lipids include,but are not limited to, N,N-distearyl-N,N-dimethylammonium bromide(DDAB), 3P—(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol(DC-Choi),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS),1,2-dileoyl-sn-3-phosphoethanolamine (DOPE),1,2-dioleoyl-3-dimethylammonium propane (DODAP),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), and 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(XTC). Additionally, commercial preparations of cationic lipids can beused, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, availablefrom GIBCO/BRL), and Lipofectamine (comprising DOSPA and DOPE, availablefrom GIBCO/BRL).

Other suitable cationic lipids are disclosed in InternationalPublication Nos. WO 09/086558, WO 09/127060, WO 10/048536, WO 10/054406,WO 10/088537, WO 10/129709, and WO 2011/153493; U.S. Patent PublicationNos. 2011/0256175, 2012/0128760, and 2012/0027803; U.S. Pat. No.8,158,601; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents ofwhich are herein incorporated by reference.

The RNA-lipid formulations of the present disclosure can comprise ahelper lipid, which can be referred to as a neutral helper lipid,non-cationic lipid, non-cationic helper lipid, anionic lipid, anionichelper lipid, or a neutral lipid. It has been found that lipidformulations, particularly cationic liposomes and lipid nanoparticleshave increased cellular uptake if helper lipids are present in theformulation. (Curr. Drug Metab. 2014; 15(9):882-92). For example, somestudies have indicated that neutral and zwitterionic lipids such as1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC),Di-Oleoyl-Phosphatidyl-Ethanoalamine (DOPE) and1,2-DiStearoyl-sn-glycero-3-PhosphoCholine (DSPC), being more fusogenic(i.e., facilitating fusion) than cationic lipids, can affect thepolymorphic features of lipid-nucleic acid complexes, promoting thetransition from a lamellar to a hexagonal phase, and thus inducingfusion and a disruption of the cellular membrane. (Nanomedicine (Lond).2014 January; 9(1):105-20). In addition, the use of helper lipids canhelp to reduce any potential detrimental effects from using manyprevalent cationic lipids such as toxicity and immunogenicity.

Non-limiting examples of non-cationic lipids suitable for lipidformulations of the present disclosure include phospholipids such aslecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylcthanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C10-C24 carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof. As a helper lipid, cholesterolincreases the spacing of the charges of the lipid layer interfacing withthe nucleic acid making the charge distribution match that of thenucleic acid more closely. (J. R. Soc. Interface. 2012 Mar. 7; 9(68):548-561). Non-limiting examples of cholesterol derivatives include polaranalogues such as 5α-cholestanol, 5α-coprostanol,cholesteryl-(2′-hydroxy)-ethyl ether, cholesteryl-(4′-hydroxy)-butylether, and 6-ketocholestanol; non-polar analogues such as 5α-cholestane,cholestenone, 5α-cholestanone, 5α-cholestanone, and cholesteryldecanoate; and mixtures thereof. In some aspects, the cholesterolderivative is a polar analogue such as cholesteryl-(4′-hydroxy)-butylether.

In some aspects, the helper lipid present in the lipid formulationcomprises or consists of a mixture of one or more phospholipids andcholesterol or a derivative thereof. In other aspects, the neutral lipidpresent in the lipid formulation comprises or consists of one or morephospholipids, e.g., a cholesterol-free lipid formulation. In yet otheraspects, the neutral lipid present in the lipid formulation comprises orconsists of cholesterol or a derivative thereof, e.g., aphospholipid-free lipid formulation.

Other examples of helper lipids include nonphosphorous containing lipidssuch as, e.g., stearylamine, dodecylamine, hexadecylamine, acetylpalmitate, glycerol ricinoleate, hexadecyl stearate, isopropylmyristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate,alkyl-aryl sulfate polyethyloxylated fatty acid amides,dioctadecyldimethyl ammonium bromide, ceramide, and sphingomyelin.

Other suitable cationic lipids include those having alternative fattyacid groups and other dialkylamino groups, including those, in which thealkyl substituents are different (e.g., N-ethyl-N-methylamino-, andN-propyl-N-ethylamino-). These lipids are part of a subcategory ofcationic lipids referred to as amino lipids. In some embodiments of thelipid formulations described herein, the cationic lipid is an aminolipid. In general, amino lipids having less saturated acyl chains aremore easily sized, particularly when the complexes must be sized belowabout 0.3 microns, for purposes of filter sterilization. Amino lipidscontaining unsaturated fatty acids with carbon chain lengths in therange of C14 to C22 may be used. Other scaffolds can also be used toseparate the amino group and the fatty acid or fatty alkyl portion ofthe amino lipid.

In some embodiments, the lipid formulation comprises the cationic lipidwith Formula I according to the patent application PCT/EP2017/064066. Inthis context, the disclosure of PCT/EP2017/064066 is also incorporatedherein by reference.

In some embodiments, amino or cationic lipids of the present disclosureare ionizable and have at least one protonatable or deprotonatablegroup, such that the lipid is positively charged at a pH1 at or belowphysiological pH (e.g., pH 7.4), and neutral at a second pH, preferablyat or above physiological pH. Of course, it will be understood that theaddition or removal of protons as a function of pH is an equilibriumprocess, and that the reference to a charged or a neutral lipid refersto the nature of the predominant species and does not require that allof the lipid be present in the charged or neutral form. Lipids that havemore than one protonatable or deprotonatable group, or which arezwitterionic, are not excluded from use in the disclosure. In certainembodiments, the protonatable lipids have a pKa of the protonatablegroup in the range of about 4 to about 11. In some embodiments, theionizable cationic lipid has a pKa of about 5 to about 7. In someembodiments, the pKa of an ionizable cationic lipid is about 6 to about7.

In some embodiments, the lipid formulation comprises an ionizablecationic lipid of Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein R5 andR6 are each independently selected from the group consisting of a linearor branched C1-C31 alkyl, C2-C31 alkenyl or C2-C31 alkynyl andcholesteryl; L5 and L6 are each independently selected from the groupconsisting of a linear C1-C20 alkyl and C2-C20 alkenyl; X5 is —C(O)O—,whereby —C(O)O—R6 is formed or —OC(O)— whereby —OC(O)—R6 is formed; X6is —C(O)O— whereby —C(O)O—R5 is formed or —OC(O)— whereby —OC(O)—R5 isformed; X7 is S or O; L7 is absent or lower alkyl; R4 is a linear orbranched C1-C6 alkyl; and R7 and R8 are each independently selected fromthe group consisting of a hydrogen and a linear or branched C1-C6 alkyl.

In some embodiments, X7 is S.

In some embodiments, X5 is —C(O)O—, whereby —C(O)O—R6 is formed and X6is —C(O)O— whereby —C(O)O—R5 is formed.

In some embodiments, R7 and R8 are each independently selected from thegroup consisting of methyl, ethyl and isopropyl.

In some embodiments, L5 and L6 are each independently a C1-C10 alkyl. Insome embodiments, L5 is C1-C3 alkyl, and L6 is C1-C5 alkyl. In someembodiments, L6 is C1-C2 alkyl. In some embodiments, L5 and L6 are eacha linear C7 alkyl. In some embodiments, L5 and L6 are each a linear C9alkyl.

In some embodiments, R5 and R6 are each independently an alkenyl. Insome embodiments, R6 is alkenyl. In some embodiments, R6 is C2-C9alkenyl. In some embodiments, the alkenyl comprises a single doublebond. In some embodiments, R5 and R6 are each alkyl. In someembodiments, R5 is a branched alkyl. In some embodiments, R5 and R6 areeach independently selected from the group consisting of a C9 alkyl, C9alkenyl and C9 alkynyl. In some embodiments, R5 and R6 are eachindependently selected from the group consisting of a C11 alkyl, C11alkenyl and C11 alkynyl. In some embodiments, R5 and R6 are eachindependently selected from the group consisting of a C7 alkyl, C7alkenyl and C7 alkynyl. In some embodiments, R5 is —CH((CH2)pCH3)2 or—CH((CH2)pCH3)((CH2)p-1CH3), wherein p is 4-8. In some embodiments, p is5 and L5 is a C1-C3 alkyl. In some embodiments, p is 6 and L5 is a C3alkyl. In some embodiments, p is 7. In some embodiments, p is 8 and L5is a C1-C3 alkyl. In some embodiments, R5 consists of—CH((CH2)pCH3)((CH2)p-1CH3), wherein p is 7 or 8.

In some embodiments, R4 is ethylene or propylene. In some embodiments,R4 is n-propylene or isobutylene.

In some embodiments, L7 is absent, R4 is ethylene, X7 is S and R7 and R8are each methyl. In some embodiments, L7 is absent, R4 is n-propylene,X7 is S and R7 and R8 are each methyl. In some embodiments, L7 isabsent, R4 is ethylene, X7 is S and R7 and R8 are each ethyl.

In some embodiments, X7 is S, X5 is —C(O)O—, whereby —C(O)O—R6 isformed, X6 is —C(O)O— whereby —C(O)O—R5 is formed, L5 and L6 are eachindependently a linear C3-C7 alkyl, L7 is absent, R5 is —CH((CH2)pCH3)2,and R6 is C7-C12 alkenyl. In some further embodiments, p is 6 and R6 isC9 alkenyl.

In some embodiments, the lipid formulation can comprise an ionizablecationic lipid selected from the group consisting of LIPID #1 to LIPID#8:

TABLE 6 LIPID # STRUCTURE 1

2

3

4

5

6

7

8

In some embodiments, the lipid formulation comprises an ionizablecationic lipid having a structure selected from

or a pharmaceutically acceptable salt thereof.

In some preferred embodiments, the lipid formulation comprises anionizable cationic lipid having the structure

or a pharmaceutically acceptable salt thereof.

In embodiments, any one or more lipids recited herein may be expresslyexcluded.

In some aspects, the helper lipid comprises from about 2 mol % to about20 mol %, from about 3 mol % to about 18 mol %, from about 4 mol % toabout 16 mol %, about 5 mol % to about 14 mol %, from about 6 mol % toabout 12 mol %, from about 5 mol % to about 10 mol %, from about 5 mol %to about 9 mol %, or about 2 mol %, about 3 mol %, about 4 mol %, about5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %,about 10 mol %, about 11 mol %, or about 12 mol % (or any fractionthereof or the range therein) of the total lipid present in the lipidformulation.

The lipid portion, or the cholesterol or cholesterol derivative in thelipid formulation may comprise up to about 40 mol %, about 45 mol %,about 50 mol %, about 55 mol %, or about 60 mol % of the total lipidpresent in the lipid formulation. In some aspects, the cholesterol orcholesterol derivative comprises about 15 mol % to about 45 mol %, about20 mol % to about 40 mol %, about 25 mol % to about 35 mol %, or about28 mol % to about 35 mol %; or about 25 mol %, about 26 mol %, about 27mol %, about 28 mol %, about 29 mol %, about 30 mol %, about 31 mol %,about 32 mol %, about 33 mol %, about 34 mol %, about 35 mol %, about 36mol/a, or about 37 mol % of the total lipid present in the lipidformulation.

In specific embodiments, the lipid portion of the lipid formulation isabout 35 mol % to about 42 mol % cholesterol.

In some aspects, the phospholipid component in the mixture may comprisefrom about 2 mol % to about 20 mol %, from about 3 mol % to about 18 mol%, from about 4 mol % to about 16 mol %, about 5 mol % to about 14 mol%, from about 6 mol % to about 12 mol %, from about 5 mol % to about 10mol %, from about 5 mol % to about 9 mol %, or about 2 mol %, about 3mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, or about 12 mol% (or any fraction thereof or the range therein) of the total lipidpresent in the lipid formulation.

In certain embodiments, the lipid portion of the lipid formulationcomprises about, but is not necessarily limited to, 40 mol % to about 60mol % of the ionizable cationic lipid, about 4 mol % to about 16 mol %DSPC, about 30 mol % to about 47 mol % cholesterol, and about 0.5 mol %to about 3 mol % PEG2000-DMG.

In certain embodiments, the lipid portion of the lipid formulation maycomprise, but is not necessarily limited to, about 42 mol % to about 58mol % of the ionizable cationic lipid, about 6 mol % to about 14 mol %DSPC, about 32 mol % to about 44 mol % cholesterol, and about 1 mol % toabout 2 mol % PEG2000-DMG.

In certain embodiments, the lipid portion of the lipid formulation maycomprise, but is not necessarily limited to, about 45 mol % to about 55mol % of the ionizable cationic lipid, about 8 mol % to about 12 mol %DSPC, about 35 mol % to about 42 mol % cholesterol, and about 1.25 mol %to about 1.75 mol % PEG2000-DMG.

The percentage of helper lipid present in the lipid formulation is atarget amount, and the actual amount of helper lipid present in theformulation may vary, for example, by ±5 mol %.

A lipid formulation that includes a cationic lipid compound or ionizablecationic lipid compound may be on a molar basis about 30-70% cationiclipid compound, about 25-40% cholesterol, about 2-15% helper lipid, andabout 0.5-5% of a polyethylene glycol (PEG) lipid, wherein the percentis of the total lipid present in the formulation. In some aspects, thecomposition is about 40-65% cationic lipid compound, about 25-35%cholesterol, about 3-9% helper lipid, and about 0.5-3% of a PEG-lipid,wherein the percent is of the total lipid present in the formulation.

The formulation may be a lipid particle formulation, for examplecontaining 8-30% nucleic acid compound, 5-30% helper lipid, and 0-20%cholesterol; 4-25% cationic lipid, 4-25% helper lipid, 2-25%cholesterol, 10-35% cholesterol-PEG, and 5% cholesterol-amine; or 2-30%cationic lipid, 2-30% helper lipid, 1-15% cholesterol, 2-35%cholesterol-PEG, and 1-20% cholesterol-amine; or up to 90% cationiclipid and 2-10% helper lipids, or even 100% cationic lipid.

Lipid Conjugates

The lipid formulations described herein may further comprise a lipidconjugate. The conjugated lipid is useful in that it prevents theaggregation of particles. Suitable conjugated lipids include, but arenot limited to, PEG-lipid conjugates, cationic-polymer-lipid conjugates,and mixtures thereof. Furthermore, lipid delivery vehicles can be usedfor specific targeting by attaching ligands (e.g., antibodies, peptides,and carbohydrates) to its surface or to the terminal end of the attachedPEG chains (Front Pharmacol. 2015 Dec. 1; 6:286).

In some aspects, the lipid conjugate is a PEG-lipid. The inclusion ofpolyethylene glycol (PEG) in a lipid formulation as a coating or surfaceligand, a technique referred to as PEGylation, helps to protectnanoparticles from the immune system and their escape from RES uptake(Nanomedicine (Lond). 2011 June; 6(4):715-28). PEGylation has been usedto stabilize lipid formulations and their payloads through physical,chemical, and biological mechanisms. Detergent-like PEG lipids (e.g.,PEG-DSPE) can enter the lipid formulation to form a hydrated layer andsteric barrier on the surface. Based on the degree of PEGylation, thesurface layer can be generally divided into two types, brush-like andmushroom-like layers. For PEG-DSPE-stabilized formulations, PEG willtake on the mushroom conformation at a low degree of PEGylation (usuallyless than 5 mol %) and will shift to brush conformation as the contentof PEG-DSPE is increased past a certain level (Journal of Nanomaterials.2011; 2011:12). PEGylation leads to a significant increase in thecirculation half-life of lipid formulations (Annu. Rev. Biomed. Eng.2011 Aug. 15; 13( ):507-30; J. Control Release. 2010 Aug. 3;145(3):178-81).

Examples of PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG),methoxypolyethyleneglycol (PEG-DMG or PEG2000-DMG), PEG coupled tophospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugatedto ceramides, PEG conjugated to cholesterol or a derivative thereof, andmixtures thereof.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights and include the following: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS),monomethoxypolyethylene glycol-amine (MePEG-NH2),monomethoxypolyethylene glycol-tresylate (MePEG-TRES),monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as wellas such compounds containing a terminal hydroxyl group instead of aterminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH2).

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain aspects, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons). In someaspects, the PEG moiety has an average molecular weight of about 2,000daltons or about 750 daltons. The average molecular weight may be anyvalue or subvalue within the recited ranges, including endpoints.

In certain aspects, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester-containing linker moieties and ester-containing linkermoieties. In one aspect, the linker moiety is a non-ester-containinglinker moiety. Exemplary non-ester-containing linker moieties include,but are not limited to, amido (—C(O)NH—), amino (—NR—), carbonyl(—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—), disulfide (—S—S—),ether (—O—), succinyl (—(O)CCH2CH2C(O)—), succinamidyl(—NHC(O)CH2CH2C(O)NH—), ether, as well as combinations thereof (such asa linker containing both a carbamate linker moiety and an amido linkermoiety). In one aspect, a carbamate linker is used to couple the PEG tothe lipid.

In some aspects, an ester-containing linker moiety is used to couple thePEG to the lipid. Exemplary ester-containing linker moieties include,e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters (—O—(O)POH—O—),sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available or can be isolated or synthesized usingconventional techniques known to those of skill in the art.Phosphatidylethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or di-unsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

In some aspects, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C10)conjugate, a PEG-dilauryloxypropyl (C12) conjugate, aPEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16)conjugate, or a PEG-distearyloxypropyl (C18) conjugate. In some aspects,the PEG has an average molecular weight of about 750 or about 2,000daltons. In some aspects, the terminal hydroxyl group of the PEG issubstituted with a methyl group.

In addition to the foregoing, other hydrophilic polymers can be used inplace of PEG. Examples of suitable polymers that can be used in place ofPEG include, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl,methacrylamide, polymethacrylamide, and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

In some aspects, the lipid conjugate (e.g., PEG-lipid) comprises fromabout 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %,from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % toabout 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, from about 0.9mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol %, fromabout 1 mol % to about 1.7 mol %, from about 1.2 mol % to about 1.8 mol%, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % toabout 1.6 mol %, or from about 1.4 mol % to about 1.6 mol % (or anyfraction thereof or range therein) of the total lipid present in thelipid formulation. In other embodiments, the lipid conjugate (e.g.,PEG-lipid) comprises about 0.5%/o, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%,1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%,4.5%, or 5%, (or any fraction thereof or range therein) of the totallipid present in the lipid formulation. The amount may be any value orsubvalue within the recited ranges, including endpoints.

The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipidformulations of the disclosure is a target amount, and the actual amountof lipid conjugate present in the formulation may vary, for example, by±0.5 mol %. One of ordinary skill in the art will appreciate that theconcentration of the lipid conjugate can be varied depending on thelipid conjugate employed and the rate at which the lipid formulation isto become fusogenic.

In some embodiments, the lipid formulation for any of the compositionsdescribed herein comprises a lipoplex, a liposome, a lipid nanoparticle,a polymer-based particle, an exosome, a lamellar body, a micelle, or anemulsion.

Mechanism of Action for Cellular Uptake of Lipid Formulations

In some aspects, lipid formulations for the intracellular delivery ofnucleic acids, particularly liposomes, cationic liposomes, and lipidnanoparticles, are designed for cellular uptake by penetrating targetcells through exploitation of the target cells' endocytic mechanismswhere the contents of the lipid delivery vehicle are delivered to thecytosol of the target cell. (Nucleic Acid Therapeutics, 28(3):146-157,2018). Prior to endocytosis, functionalized ligands such as PEG-lipid atthe surface of the lipid delivery vehicle are shed from the surface,which triggers internalization into the target cell. During endocytosis,some part of the plasma membrane of the cell surrounds the vector andengulfs it into a vesicle that then pinches off from the cell membrane,enters the cytosol and ultimately enters and moves through theendolysosomal pathway. For ionizable cationic lipid-containing deliveryvehicles, the increased acidity as the endosome ages results in avehicle with a strong positive charge on the surface. Interactionsbetween the delivery vehicle and the endosomal membrane then result in amembrane fusion event that leads to cytosolic delivery of the payload.For RNA payloads, the cell's own internal translation processes willthen translate the RNA into the encoded protein. The encoded protein canfurther undergo postranslational processing, including transportation toa targeted organelle or location within the cell or excretion from thecell.

By controlling the composition and concentration of the lipid conjugate,one can control the rate at which the lipid conjugate exchanges out ofthe lipid formulation and, in turn, the rate at which the lipidformulation becomes fusogenic. In addition, other variables including,e.g., pH, temperature, or ionic strength, can be used to vary and/orcontrol the rate at which the lipid formulation becomes fusogenic. Othermethods which can be used to control the rate at which the lipidformulation becomes fusogenic will become apparent to those of skill inthe art upon reading this disclosure. Also, by controlling thecomposition and concentration of the lipid conjugate, one can controlthe liposomal or lipid particle size.

Lipid Formulation Manufacture

There are many different methods for the preparation of lipidformulations comprising a nucleic acid. (Curr. Drug Metabol. 2014, 15,882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res.2012, 3, 14-20). The techniques of thin film hydration, double emulsion,reverse phase evaporation, microfluidic preparation, dual assymetriccentrifugation, ethanol injection, detergent dialysis, spontaneousvesicle formation by ethanol dilution, and encapsulation in preformedliposomes are briefly described herein.

Thin Film Hydration

In Thin Film Hydration (TFH) or the Bangham method, the lipids aredissolved in an organic solvent, then evaporated through the use of arotary evaporator leading to a thin lipid layer formation. After thelayer hydration by an aqueous buffer solution containing the compound tobe loaded, Multilamellar Vesicles (MLVs) are formed, which can bereduced in size to produce Small or Large Unilamellar vesicles (LUV andSUV) by extrusion through membranes or by the sonication of the startingMLV.

Double Emulsion

Lipid formulations can also be prepared through the Double Emulsiontechnique, which involves lipids dissolution in a water/organic solventmixture. The organic solution, containing water droplets, is mixed withan excess of aqueous medium, leading to a water-in-oil-in-water (W/O/W)double emulsion formation. After mechanical vigorous shaking, part ofthe water droplets collapse, giving Large Unilamellar Vesicles (LUVs).

Reverse Phase Evaporation

The Reverse Phase Evaporation (REV) method also allows one to achieveLUVs loaded with nucleic acid. In this technique a two-phase system isformed by phospholipids dissolution in organic solvents and aqueousbuffer. The resulting suspension is then sonicated briefly until themixture becomes a clear one-phase dispersion. The lipid formulation isachieved after the organic solvent evaporation under reduced pressure.This technique has been used to encapsulate different large and smallhydrophilic molecules including nucleic acids.

Microfluidic Preparation

The Microfluidic method, unlike other bulk techniques, gives thepossibility of controlling the lipid hydration process. The method canbe classified in continuous-flow microfluidic and droplet-basedmicrofluidic, according to the way in which the flow is manipulated. Inthe microfluidic hydrodynamic focusing (MHF) method, which operates in acontinuous flow mode, lipids are dissolved in isopropyl alcohol which ishydrodynamically focused in a microchannel cross junction between twoaqueous buffer streams. Vesicles size can be controlled by modulatingthe flow rates, thus controlling the lipids solution/buffer dilutionprocess. The method can be used for producing oligonucleotide (ON) lipidformulations by using a microfluidic device consisting of three-inletand one-outlet ports.

Dual Asymmetric Centrifugation

Dual Asymmetric Centrifugation (DAC) differs from more commoncentrifugation as it uses an additional rotation around its own verticalaxis. An efficient homogenization is achieved due to the two overlayingmovements generated: the sample is pushed outwards, as in a normalcentrifuge, and then it is pushed towards the center of the vial due tothe additional rotation. By mixing lipids and an NaCl-solution a viscousvesicular phospholipid gel (VPC) is achieved, which is then diluted toobtain a lipid formulation dispersion. The lipid formulation size can beregulated by optimizing DAC speed, lipid concentration andhomogenization time.

Ethanol Injection

The Ethanol Injection (EI) method can be used for nucleic acidencapsulation. This method provides the rapid injection of an ethanolicsolution, in which lipids are dissolved, into an aqueous mediumcontaining nucleic acids to be encapsulated, through the use of aneedle. Vesicles are spontaneously formed when the phospholipids aredispersed throughout the medium.

Detergent Dialysis

The Detergent dialysis method can be used to encapsulate nucleic acids.Briefly lipid and plasmid are solubilized in a detergent solution ofappropriate ionic strength, after removing the detergent by dialysis, astabilized lipid formulation is formed. Unencapsulated nucleic acid isthen removed by ion-exchange chromatography and empty vesicles bysucrose density gradient centrifugation. The technique is highlysensitive to the cationic lipid content and to the salt concentration ofthe dialysis buffer, and the method is also difficult to scale.

Spontaneous Vesicle Formation by Ethanol Dilution

Stable lipid formulations can also be produced through the SpontaneousVesicle Formation by Ethanol Dilution method in which a stepwise ordropwise ethanol dilution provides the instantaneous formation ofvesicles loaded with nucleic acid by the controlled addition of lipiddissolved in ethanol to a rapidly mixing aqueous buffer containing thenucleic acid.

Encapsulation in Preformed Liposomes

The entrapment of nucleic acids can also be obtained starting withpreformed liposomes through two different methods: (1) A simple mixingof cationic liposomes with nucleic acids which gives electrostaticcomplexes called “lipoplexes”, where they can be successfully used totransfect cell cultures, but are characterized by their lowencapsulation efficiency and poor performance in vivo; and (2) aliposomal destabilization, slowly adding absolute ethanol to asuspension of cationic vesicles up to a concentration of 40% v/vfollowed by the dropwise addition of nucleic acids achieving loadedvesicles; however, the two main steps characterizing the encapsulationprocess are too sensitive, and the particles have to be downsized.

Excipients

The pharmaceutical compositions disclosed herein can be formulated usingone or more excipients to: (1) increase stability; (2) increase celltransfection; (3) permit a sustained or delayed release (e.g., from adepot formulation of the polynucleotide, primary construct, or RNA); (4)alter the biodistribution (e.g., target the polynucleotide, primaryconstruct, or RNA to specific tissues or cell types); (5) increase thetranslation of encoded protein in vivo; and/or (6) alter the releaseprofile of encoded protein in vivo.

The pharmaceutical compositions described herein may be prepared by anymethod known or hereafter developed in the art of pharmacology. Ingeneral, such preparatory methods include the step of associating theactive ingredient (i.e., nucleic acid) with an excipient and/or one ormore other accessory ingredients. A pharmaceutical composition inaccordance with the present disclosure may be prepared, packaged, and/orsold in bulk, as a single unit dose, and/or as a plurality of singleunit doses.

Pharmaceutical compositions may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired.

In addition to traditional excipients such as any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, excipients of the present disclosurecan include, without limitation, liposomes, lipid nanoparticles,polymers, lipoplexes, core-shell nanoparticles, peptides, proteins,cells transfected with primary DNA construct, or RNA (e.g., fortransplantation into a subject), hyaluronidase, nanoparticle mimics andcombinations thereof.

Accordingly, the pharmaceutical compositions described herein caninclude one or more excipients, each in an amount that togetherincreases the stability of the nucleic acid in the lipid formulation,increases cell transfection by the nucleic acid, increases theexpression of the encoded protein, and/or alters the release profile ofencoded proteins. Further, the RNA of the present disclosure may beformulated using self-assembled nucleic acid nanoparticles.

Various excipients for formulating pharmaceutical compositions andtechniques for preparing the composition are known in the art (seeRemington: The Science and Practice of Pharmacy, 21st Edition, A. R.Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006;incorporated herein by reference in its entirety). The use of aconventional excipient medium may be contemplated within the scope ofthe embodiments of the present disclosure, except insofar as anyconventional excipient medium may be incompatible with a substance orits derivatives, such as by producing any undesirable biological effector otherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition.

The pharmaceutical compositions of this disclosure may further containas pharmaceutically acceptable carriers substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, and wetting agents, for example,sodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, sorbitan monolaurate, triethanolamine oleate, andmixtures thereof. For solid compositions, conventional nontoxicpharmaceutically acceptable carriers can be used which include, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

In certain embodiments of the disclosure, the RNA-lipid formulation maybe administered in a time release formulation, for example in acomposition which includes a slow release polymer. The active agent canbe prepared with carriers that will protect against rapid release, forexample a controlled release vehicle such as a polymer,microencapsulated delivery system, or a bioadhesive gel. Prolongeddelivery of the RNA, in various compositions of the disclosure can bebrought about by including in the composition agents that delayabsorption, for example, aluminum monostearate hydrogels and gelatin.

Methods of Inducing Immune Responses

Provided herein, in some embodiments, are methods of inducing an immuneresponse in a subject. Any type of immune response can be induced usingthe methods provided herein, including adaptive and innate immuneresponses. In one aspect, immune responses induced using the methodsprovided herein include an antibody response, a cellular immuneresponse, or both an antibody response and a cellular immune response.

Methods of inducing an immune response provided herein includeadministering to a subject an effective amount of any nucleic acidmolecule provided herein. In one aspect, methods of inducing an immuneresponse include administering to a subject an effective amount of anycomposition comprising a nucleic acid molecule and a lipid providedherein. In another aspect, methods of inducing an immune responseinclude administering to a subject an effective amount of anypharmaceutical composition comprising a nucleic acid molecule and alipid formulation provided herein. In some aspects, nucleic acidmolecules, compositions, and pharmaceutical composition provided hereare vaccines that can elicit a protective or a therapeutic immuneresponse, for example.

As used herein, the term “subject” refers to any individual or patienton which the methods disclosed herein are performed. The term “subject”can be used interchangeably with the term “individual” or “patient.” Thesubject can be a human, although the subject may be an animal, as willbe appreciated by those in the art. Thus, other animals, includingmammals such as rodents (including mice, rats, hamsters and guineapigs), cats, dogs, rabbits, farm animals including cows, horses, goats,sheep, pigs, etc., and primates (including monkeys, chimpanzees,orangutans and gorillas) are included within the definition of subject.As used herein, the term “effective amount” or “therapeuticallyeffective amount” refers to that amount of a nucleic acid molecule,composition, or pharmaceutical composition described herein that issufficient to effect the intended application, including but not limitedto inducing an immune response and/or disease treatment, as definedherein. The therapeutically effective amount may vary depending upon theintended application (e.g., inducing an immune response, treatment,application in vivo), or the subject or patient and disease conditionbeing treated, e.g., the weight and age of the subject, the species, theseverity of the disease condition, the manner of administration and thelike, which can readily be determined by one of ordinary skill in theart. The term also applies to a dose that will induce a particularresponse in a target cell. The specific dose will vary depending on theparticular nucleic acid molecule, composition, or pharmaceuticalcomposition chosen, the dosing regimen to be followed, whether it isadministered in combination with other compounds, timing ofadministration, the tissue to which it is administered, and the physicaldelivery system in which it is carried.

Exemplary doses of nucleic acid molecules that can be administeredinclude about 0.01 μg, about 0.02 μg, about 0.03 μg, about 0.04 μg,about 0.05 μg, about 0.06 μg, about 0.07 μg, about 0.08 μg, about 0.09μg, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1.0μg, about 1.5 μg, about 2.0 μg, about 2.5 μg, about 3.0 μg, about 3.5μg, about 4.0 μg, about 4.5 μg, about 5.0 μg, about 5.5 μg, about 6.0μg, about 6.5 μg, about 7.0 μg, about 7.5 μg, about 8.0 μg, about 8.5μg, about 9.0 μg, about 9.5 μg, about 10 μg, about 11 μg, about 12 μg,about 13μ, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg,about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about29 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg,about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about80 μg, about 85 μg, about 90 μg, about 95 μg, about 100 μg, about 125μg, about 150 μg, about 175 μg, about 200 μg, about 250 μg, about 300μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600μg, about 700 μg, about 800 μg, about 900 μg, about 1,000 μg, or more,and any number or range in between. In one aspect, the nucleic acidmolecules are RNA molecules. In another aspect, the nucleic acidmolecules are DNA molecules. Nucleic acid molecules can have a unitdosage comprising about 0.01 μg to about 1,000 μg or more nucleic acidin a single dose.

In some aspects, compositions provided herein that can be administeredinclude about 0.01 μg, about 0.02 μg, about 0.03 μg, about 0.04 μg,about 0.05 μg, about 0.06 μg, about 0.07 μg, about 0.08 μg, about 0.09μg, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1.0μg, about 1.5 μg, about 2.0 μg, about 2.5 μg, about 3.0 μg, about 3.5μg, about 4.0 μg, about 4.5 μg, about 5.0 μg, about 5.5 μg, about 6.0μg, about 6.5 μg, about 7.0 μg, about 7.5 μg, about 8.0 μg, about 8.5μg, about 9.0 μg, about 9.5 μg, about 10 μg, about 11 μg, about 12 μg,about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg,about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about29 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg,about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about80 μg, about 85 μg, about 90 μg, about 95 μg, about 100 μg, about 125μg, about 150 μg, about 175 μg, about 200 μg, about 250 μg, about 300μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600μg, about 700 μg, about 800 μg, about 900 μg, about 1,000 μg, or more,and any number or range in between, nucleic acid and lipid. In otheraspects, pharmaceutical compositions provided herein that can beadministered include about 0.01 μg, about 0.02 μg, about 0.03 μg, about0.04 μg, about 0.05 μg, about 0.06 μg, about 0.07 μg, about 0.08 μg,about 0.09 μg, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg,about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg,about 1.0 μg, about 1.5 μg, about 2.0 μg, about 2.5 μg, about 3.0 μg,about 3.5 μg, about 4.0 μg, about 4.5 μg, about 5.0 μg, about 5.5 μg,about 6.0 μg, about 6.5 μg, about 7.0 μg, about 7.5 μg, about 8.0 μg,about 8.5 μg, about 9.0 μg, about 9.5 μg, about 10 μg, about 11 μg,about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg,about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about28 μg, about 29 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg,about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, about 100 μg,about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 250 μg,about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg,about 600 μg, about 700 μg, about 800 μg, about 900 μg, about 1,000 μg,or more, and any number or range in between, nucleic acid and lipidformulation.

In one aspect, compositions provided herein can have a unit dosagecomprising about 0.01 μg to about 1,000 μg or more nucleic acid andlipid in a single dose. In another aspect, pharmaceutical compositionsprovided herein can have a unit dosage comprising about 0.01 μg to about1,000 μg or more nucleic acid and lipid formulation in a single dose. Avaccine unit dosage can correspond to the unit dosage of nucleic acidmolecules, compositions, or pharmaceutical compositions provided hereinand that can be administered to a subject. In one aspect, vaccinecompositions of the instant disclosure have a unit dosage comprisingabout 0.01 μg to about 1,000 μg or more nucleic acid and lipidformulation in a single dose. In another aspect, vaccine compositions ofthe instant disclosure have a unit dosage comprising about 0.01 μg toabout 50 μg nucleic acid and lipid formulation in a single dose. In yetanother aspect, vaccine compositions of the instant disclosure have aunit dosage comprising about 0.2 μg to about 20 μg nucleic acid andlipid formulation in a single dose.

A dosage form of the composition of this disclosure can be solid, whichcan be reconstituted in a liquid prior to administration. The solid canbe administered as a powder. The solid can be in the form of a capsule,tablet, or gel. In some embodiments, the pharmaceutical compositioncomprises a nucleic acid lipid formulation that has been lyophilized. Insome embodiments, the lyophilized composition may comprise one or morelyoprotectants, such as, including but not necessarily limited to,glucose, trehalose, sucrose, maltose, lactose, mannitol, inositol,hydroxypropyl-p-cyclodextrin, and/or polyethylene glycol. In someembodiments, the lyophilized composition comprises a poloxamer,potassium sorbate, sucrose, or any combination thereof. In specificembodiments, the poloxamer is poloxamer 188. In some embodiments, thelyophilized compositions described herein may comprise about 0.01 toabout 1.0% w/w of a poloxamer. In some embodiments, the lyophilizedcompositions described herein may comprise about 1.0 to about 5.0% w/wof potassium sorbate. The percentages may be any value or subvaluewithin the recited ranges, including endpoints.

In some embodiments, the lyophilized composition may comprise about 0.01to about 1.0% w/w of the nucleic acid molecule. In some embodiments, thecomposition may comprise about 1.0 to about 5.0% w/w lipids. In someembodiments, the composition may comprise about 0.5 to about 2.5% w/w ofTRIS buffer. In some embodiments, the composition may comprise about0.75 to about 2.75% w/w of NaCl. In some embodiments, the compositionmay comprise about 85 to about 95% w/w of a sugar. The percentages maybe any value or subvalue within the recited ranges, including endpoints.

In a preferred embodiment, the dosage form of the pharmaceuticalcompositions described herein can be a liquid suspension ofself-replicating RNA lipid nanoparticles described herein. In someembodiments, the liquid suspension is in a buffered solution. In someembodiments, the buffered solution comprises a buffer selected from thegroup consisting of HEPES, MOPS, TES, and TRIS. In some embodiments, thebuffer has a pH of about 7.4. In some preferred embodiments, the bufferis HEPES. In some further embodiments, the buffered solution furthercomprises a cryoprotectant. In some embodiments, the cryoprotectant isselected from a sugar and glycerol or a combination of a sugar andglycerol. In some embodiments, the sugar is a dimeric sugar. In someembodiments, the sugar is sucrose. In some preferred embodiments, thebuffer comprises HEPES, sucrose, and glycerol at a pH of 7.4. In certainembodiments, the composition comprises a HEPES, MOPS, TES, or TRISbuffer at a pH of about 7.0 to about 8.5. In some embodiments, theHEPES, MOPS, TES, or TRIS buffer may at a concentration ranging from 7mg/ml to about 15 mg/ml. The pH or concentration may be any value orsubvalue within the recited ranges, including endpoints.

In some embodiments, the suspension is frozen during storage and thawedprior to administration. In some embodiments, the suspension is frozenat a temperature below about 70° C. In some embodiments, the suspensionis diluted with sterile water during intravenous administration. In someembodiments, intravenous administration comprises diluting thesuspension with about 2 volumes to about 6 volumes of sterile water. Insome embodiments, the suspension comprises about 0.1 mg to about 3.0 mgself-replicating RNA/mL, about 15 mg/mL to about 25 mg/mL of anionizable cationic lipid, about 0.5 mg/mL to about 2.5 mg/mL of aPEG-lipid, about 1.8 mg/mL to about 3.5 mg/mL of a helper lipid, about4.5 mg/mL to about 7.5 mg/mL of a cholesterol, about 7 mg/mL to about 15mg/mL of a buffer, about 2.0 mg/mL to about 4.0 mg/mL of NaCl, about 70mg/mL to about 110 mg/mL of sucrose, and about 50 mg/mL to about 70mg/mL of glycerol. In some embodiments, a lyophilized self-replicatingRNA-lipid nanoparticle formulation can be resuspended in a buffer asdescribed herein.

In some embodiments, the compositions of the disclosure are administeredto a subject such that a self-replicating RNA concentration of at leastabout 0.05 mg/kg, at least about 0.1 mg/kg, at least about 0.5 mg/kg, atleast about 1.0 mg/kg, at least about 2.0 mg/kg, at least about 3.0mg/kg, at least about 4.0 mg/kg, at least about 5.0 mg/kg of body weightis administered in a single dose or as part of single treatment cycle.In some embodiments, the compositions of the disclosure are administeredto a subject such that a total amount of at least about 0.1 mg, at leastabout 0.5 mg, at least about 1.0 mg, at least about 2.0 mg, at leastabout 3.0 mg, at least about 4.0 mg, at least about 5.0 mg, at leastabout 6.0 mg, at least about 7.0 mg, at least about 8.0 mg, at leastabout 9.0 mg, at least about 10 mg, at least about 15 mg, at least about20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg,at least about 40 mg, at least about 45 mg, at least about 50 mg, atleast about 55 mg, at least about 60 mg, at least about 65 mg, at leastabout 70 mg, at least about 75 mg, at least about 80 mg, at least about85 mg, at least about 90 mg, at least about 95 mg, at least about 100mg, at least about 105 mg, at least about 110 mg, at least about 115 mg,at least about 120 mg, or at least about 125 mg self-replicating RNA isadministered in one or more doses up to a maximum dose of about 300 mg,about 350 mg, about 400 mg, about 450 mg, or about 500 mgself-replicating RNA.

Any route of administration can be included in methods provided herein.In some aspects, nucleic acid molecules, compositions, andpharmaceutical compositions provided herein are administeredintramuscularly, subcutaneously, intradermally, transdermally,intranasally, orally, sublingually, intravenously, intraperitoneally,topically, by aerosol, or by a pulmonary route, such as by inhalation orby nebulization, for example. In some embodiments, the pharmaceuticalcompositions described are administered systemically. Suitable routes ofadministration include, for example, oral, rectal, vaginal,transmucosal, pulmonary including intratracheal or inhaled, orintestinal administration; parenteral delivery, including intradermal,transdermal (topical), intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, or intranasal. In particular embodiments,the intramuscular administration is to a muscle selected from the groupconsisting of skeletal muscle, smooth muscle and cardiac muscle. In someembodiments, the pharmaceutical composition is administeredintravenously.

Pharmaceutical compositions may be administered to any desired tissue.In some embodiments, the self-replicating RNA delivered is expressed ina tissue different from the tissue in which the lipid formulation orpharmaceutical composition was administered. In preferred embodiments,self-replicating RNA is delivered and expressed in the liver.

In other aspects, nucleic acid molecules, compositions, andpharmaceutical compositions provided herein are administeredintramuscularly.

In some aspects, the subject in which an immune response is induced is ahealthy subject. As used herein, the term “healthy subject” refers to asubject not having a condition or disease, including an infectiousdisease or cancer, for example, or not having a condition or diseaseagainst which an immune response is induced. Accordingly, in someaspects, a nucleic acid molecule, composition, or pharmaceuticalcomposition provided herein is administered prophylactically to preventan infectious disease or cancer, for example. In other aspects, thesubject in which an immune response is induced has cancer. The subjectmay suffer from any cancer or have any tumor, including solid and liquidtumors. In one aspect, the cancer is kidney cancer, renal cancer,urinary bladder cancer, prostate cancer, uterine cancer, breast cancer,cervical cancer, ovarian cancer, lung cancer, liver cancer, stomachcancer, colon cancer, rectal cancer, oral cavity cancer, pharynx cancer,pancreatic cancer, thyroid cancer, melanoma, skin cancer, head and neckcancer, brain cancer, hematopoietic cancer, leukemia, lymphoma, bonecancer, or sarcoma. Accordingly, a nucleic acid molecule, composition,or pharmaceutical composition provided herein can be administeredtherapeutically, i.e., to treat a condition or disease, such as cancer,after the onset of the condition or disease.

As used herein, the terms “treat,” “treatment,” “therapy,”“therapeutic,” and the like refer to obtaining a desired pharmacologicand/or physiologic effect, including, but not limited to, alleviating,delaying or slowing the progression, reducing the effects or symptoms,preventing onset, inhibiting, ameliorating the onset of a diseases ordisorder, obtaining a beneficial or desired result with respect to adisease, disorder, or medical condition, such as a therapeutic benefitand/or a prophylactic benefit. “Treatment,” as used herein, includes anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject,including a subject which is predisposed to the disease or at risk ofacquiring the disease but has not yet been diagnosed as having it; (b)inhibiting the disease, i.e., arresting its development; and (c)relieving the disease, i.e., causing regression of the disease. Atherapeutic benefit includes eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the subject, notwithstanding that thesubject may still be afflicted with the underlying disorder. In someaspects, for prophylactic benefit, treatment or compositions fortreatment, including pharmaceutical compositions, are administered to asubject at risk of developing a particular disease, or to a subjectreporting one or more of the physiological symptoms of a disease, eventhough a diagnosis of this disease may not have been made. The methodsof the present disclosure may be used with any mammal or other animal.In some aspects, treatment results in a decrease or cessation ofsymptoms. A prophylactic effect includes delaying or eliminating theappearance of a disease or condition, delaying or eliminating the onsetof symptoms of a disease or condition, slowing, halting, or reversingthe progression of a disease or condition, or any combination thereof.

Nucleic acid molecules, compositions, and pharmaceutical compositionsprovided herein can be administered once or multiple times. Accordingly,nucleic acid molecules, compositions, and pharmaceutical compositionsprovided herein can be administered one, two, three, four, five, six,seven, eight, nine, ten, or more times. Timing between two or moreadministrations can be one week, two weeks, three weeks, four weeks,five weeks, six weeks, seven weeks, eight weeks, nine weeks, weeks, tenweeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52weeks, or more weeks, and any number or range in between. In someaspects, timing between two or more administrations is one month, twomonths, three months, four months, five months, six months, sevenmonths, eight months, nine months, ten months, 11 months, 12 months, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months, 24 months, or moremonths, and any number or range in between. In other aspects, timingbetween two or more administrations can be one year, two years, threeyears, four years, five years, six years, seven years, eight years, nineyears, ten years, or more years, and any number or range in between,Timing between the first and any subsequent administration can be thesame or different. In one aspect, nucleic acid molecules, compositions,or pharmaceutical compositions provided herein are administered once.

More than one nucleic acid molecule, composition, or pharmaceuticalcomposition can be administered in the methods provided herein. In oneaspect, two or more nucleic acid molecules, compositions, orpharmaceutical compositions provided herein are administeredsimultaneously. In another aspect, two or more nucleic acid molecules,compositions, or pharmaceutical compositions provided herein areadministered sequentially. Simultaneous and sequential administrationscan include any number and any combination of nucleic acid molecules,compositions, or pharmaceutical compositions provided herein. Multiplenucleic acid molecules, compositions, or pharmaceutical compositionsthat are administered together or sequentially can include transgenesencoding different antigenic proteins or fragments thereof. In thismanner, immune responses against different antigenic targets can beinduced. Two, three, four, five, six, seven, eight, nine, ten, or morenucleic acid molecules, compositions, or pharmaceutical compositionsincluding transgenes encoding different antigenic proteins or fragmentsthereof can be administered simultaneously or sequentially. Anycombination of nucleic acid molecules, compositions, and pharmaceuticalcompositions including any combination of transgenes can be administeredsimultaneously or sequentially. In some aspects, administration issimultaneous. In other aspects, administration is sequential. Timingbetween two or more administrations can be one week, two weeks, threeweeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nineweeks, weeks, ten weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50weeks, 51 weeks, 52 weeks, or more weeks, and any number or range inbetween. In some aspects, timing between two or more administrations isone month, two months, three months, four months, five months, sixmonths, seven months, eight months, nine months, ten months, 11 months,12 months, 13 months, 14 months, 15 months, months, 16 months, 17months, 18 months, 19 months, 20 months, 21 months, 22 months, 23months, 24 months, or more months, and any number or range in between.In other aspects, timing between two or more administrations can be oneyear, two years, three years, four years, five years, six years, sevenyears, eight years, nine years, ten years, or more years, and any numberor range in between, Timing between the first and any subsequentadministration can be the same or different. Nucleic acid molecules,compositions, and pharmaceutical compositions provided herein can beadministered with any other vaccine or treatment.

Following administration of the composition to the subject, the proteinproduct encoded by the self-replicating RNA of the disclosure (e.g., anantigen) is detectable in the target tissues for at least about one toseven days or longer. The amount of protein product necessary to achievea therapeutic effect will vary depending on antibody titer necessary togenerate an immunity to COVID-19 in the patient. For example, theprotein product may be detectable in the target tissues at aconcentration (e.g., a therapeutic concentration) of at least about0.025-1.5 μg/ml (e.g., at least about 0.050 μg/ml, at least about 0.075μg/ml, at least about 0.1 μg/ml, at least about 0.2 μg/ml, at leastabout 0.3 pig/ml, at least about 0.4 pig/ml, at least about 0.5 μg/ml,at least about 0.6 μg/ml, at least about 0.7 μg/ml, at least about 0.8μg/ml, at least about 0.9 μg/ml, at least about 1.0 μg/ml, at leastabout 1.1 μg/ml, at least about 1.2 μg/ml, at least about 1.3 μg/ml, atleast about 1.4 μg/ml, or at least about 1.5 μg/ml), for at least about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 days or longerfollowing administration of the composition to the subject.

In some embodiments, the composition described herein may beadministered one time. In some embodiments, the composition describedherein may be administered two times.

In some embodiments, the composition may be administered in the form ofa booster dose, to a subject who was previously vaccinated againstcoronavirus.

In some embodiments, a pharmaceutical composition of the presentdisclosure is administered to a subject once per month. In someembodiments, a pharmaceutical composition of the present disclosure isadministered to a subject twice per month. In some embodiments, apharmaceutical composition of the present disclosure is administered toa subject three times per month. In some embodiments, a pharmaceuticalcomposition of the present disclosure is administered to a subject fourtimes per month.

Alternatively, the compositions of the present disclosure may beadministered in a local rather than systemic manner, for example, viainjection of the pharmaceutical composition directly into a targetedtissue, preferably in a depot or sustained release formulation. Localdelivery can be affected in various ways, depending on the tissue to betargeted. For example, aerosols containing compositions of the presentdisclosure can be inhaled (for nasal, tracheal, or bronchial delivery);compositions of the present disclosure can be injected into the site ofinjury, disease manifestation, or pain, for example; compositions can beprovided in lozenges for oral, tracheal, or esophageal application; canbe supplied in liquid, tablet or capsule form for administration to thestomach or intestines, can be supplied in suppository form for rectal orvaginal application; or can even be delivered to the eye by use ofcreams, drops, or even injection. Formulations containing compositionsof the present disclosure complexed with therapeutic molecules orligands can even be surgically administered, for example in associationwith a polymer or other structure or substance that can allow thecompositions to diffuse from the site of implantation to surroundingcells. Alternatively, they can be applied surgically without the use ofpolymers or supports.

Combinations

The self-replicating RNA, formulations thereof, or encoded proteinsdescribed herein may be used in combination with one or more othertherapeutic, prophylactic, diagnostic, or imaging agents. By “incombination with,” it is not intended to imply that the agents must beadministered at the same time and/or formulated for delivery together,although these methods of delivery are within the scope of the presentdisclosure. Compositions can be administered concurrently with, priorto, or subsequent to, one or more other desired therapeutics or medicalprocedures. In general, each agent will be administered at a dose and/oron a time schedule determined for that agent. Preferably, the methods oftreatment of the present disclosure encompass the delivery ofpharmaceutical, prophylactic, diagnostic, or imaging compositions incombination with agents that may improve their bioavailability, reduceand/or modify their metabolism, inhibit their excretion, and/or modifytheir distribution within the body. As a non-limiting example, aself-replicating RNA of the disclosure may be used in combination with apharmaceutical agent for immunizing or vaccinating a subject. Ingeneral, it is expected that agents utilized in combination with thepresently disclosed self-replicating RNA and formulations thereof beutilized at levels that do not exceed the levels at which they areutilized individually. In some embodiments, the levels utilized incombination will be lower than those utilized individually. In oneembodiment, the combinations, each or together may be administeredaccording to the split dosing regimens as are known in the art.

Ranges

Throughout this disclosure, various aspects can be presented in rangeformat. It should be understood that any description in range format ismerely for convenience and brevity and not meant to be limiting.Accordingly, the description of a range should be considered to havespecifically disclosed all possible subranges as well as individualnumerical values within that range. For example, description of a rangesuch as from 1 to 6 should be considered to have specifically disclosedsubranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6, etc., as well as individual numbers withinthat range, for example 1, 2, 2.1, 2.2, 2.5, 3, 4, 4.75, 4.8, 4.85,4.95, 5, 5.5, 5.75, 5.9, 5.00, and 6. This applies to a range of anybreadth.

Example 1

This example describes a comparison of design and expression of mRNA andself-replicating RNA (STARR™) platforms.

Both mRNA and STARR™ vaccine constructs were designed to encode thefull-length SARS-CoV-2 S protein (1273 aa), with the STARR™self-replicating RNA additionally encoding for the Venezuelan equineencephalitis virus (VEEV) replicase genes (FIG. 1A; STARR™ vaccineconstruct corresponding to an RNA having a sequence of SEQ ID NO:125,with U in place of T, referred to herein as “STARR™ SARS-CoV-2 RNA”;mRNA corresponding to a sequence of SEQ ID NO:126, with U in place of Tand including a tobacco etch virus (TEV) 5′ UTR, a Xenopus beta-globin(Xbg) 3′ UTR, and a codon-optimized open reading frame encoding theSARS-CoV-2 glycoprotein). The characteristics of these differentconstructs was studied first. Constructs were encapsulated in the sameLNP composition. Briefly, RNA constructs were encapsulated into lipidnanoparticles (LNPs) that included four lipid excipients (an ionizablecationic lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),cholesterol, and PEG2000-DMG) dispersed in HEPES buffer (pH 8.0)containing sodium chloride and the cryoprotectants sucrose and glycerol.The ionizable cationic lipid had the following structure:

Despite differences in RNA lengths of mRNA and the STARR™ SARS-CoV-2 RNAconstruct, the LNP diameter, polydispersity index and RNA trappingefficiency were similar (FIG. 1B). In vitro expression of the mRNAvaccine and the STARR™ SARS-CoV-2 RNA construct were confirmed in celllysate 24 hours post-transfection through positive western blotdetection of the S protein (FIG. 1C). It was also observed that bothmRNA and STARR™ vaccines expressed a mixture of full-length S proteinand cleaved S protein, i.e., S was cleaved into S1 and S2 transmembraneand cytoplasmic membrane domains (FIG. 1C). In vivo protein expressionof the two RNA platforms in BALB/c mice was compared by using mRNA andSTARR™ constructs that expressed a luciferase reporter (FIG. 1D).Animals injected with the mRNA vaccine construct showed high in vivoluciferase expression at day 1, although the expression levels declinedover time. In contrast, luciferase expression in STARR™ injected miceshowed sustained or even increased signals, apart from those given the0.2 μg dose, until day 7 post-inoculation (FIG. 1D).

These data show that dose-for-dose, antigen expression was moreprolonged with the STARR™ compared to the mRNA vaccine.

Example 2

This example describes immune gene expression following the STARR™construct and mRNA vaccination.

C57BL/6J mice were inoculated with STARR™ SARS-CoV-2 RNA (encoding theSARS-CoV-2 glycoprotein as described above (Example 1)) and mRNAvaccines at 0.2 μg, 2 μg and 10 μg doses or PBS control. No significantmean loss in animal weight occurred over the first 4 days, except forthose that received 10 μg of STARR™ SARS-CoV-2 RNA (FIG. 2A). However,apart from weight loss, there were few other clinical signs as indicatedby the minimal differences in clinical scores. Both weight and clinicalscores improved after day 3 post vaccination.

The innate immune response, such as the type-I interferon (IFN)response, has previously been shown to be associated with vaccineimmunogenicity following yellow fever vaccination, for example.Furthermore, reactive oxygen species-driven pro-inflammatory responseshave been shown to underpin systemic adverse events in yellow fevervaccination. Therefore, the expression of innate immune andpro-inflammatory genes in whole blood of C57BL/6 mice inoculated wasmeasured with either PBS, mRNA vaccine, or the STARR™ SARS-CoV-2 RNAconstruct. Genes in the type-I IFN pathway were the most highlyexpressed in animals inoculated with STARR™ SARS-CoV-2 RNA compared toeither mRNA vaccine or PBS (FIG. 2B and FIG. 8 ). By contrast, genesassociated with pro-inflammatory responses were mostly reduced inabundance following vaccination STARR™ SARS-CoV-2 RNA compared witheither mRNA vaccine or PBS (FIG. 2B and FIG. 8 ).

Since adaptive immune responses develop in germinal centers in thedraining lymph nodes, the draining lymph nodes were dissected at day 7post-inoculation (study schematic in FIG. 2A). The inguinal lymph nodesof mice inoculated with STARR™ SARS-CoV-2 RNA showed a dose-dependentincrease in weight, unlike those from mice inoculated with either mRNAvaccine or PBS; the mean weight of lymph nodes from mice given 10 μg ofSTARR™ SARS-CoV-2 RNA was significantly higher than those given theequivalent mRNA vaccine (FIG. 2C). Principal component analysis (PCA) ofimmune gene expression showed clustering of responses to each of the 3doses of STARR™ SARS-CoV-2 RNA away from the PBS control (STARR™ RNA):depicted as lower sphere in FIG. 2D, smallest sphere in FIG. 2E, andlower sphere in FIG. 2F; PBS control: depicted as upper sphere in FIG.2D, lower elongated and narrow sphere in FIG. 2E, and upper sphere inFIG. 2F) indicating clear differences in immune gene expression betweenSTARR™ SARS-CoV-2 RNA vaccinated and placebo groups. These trends werealso dissimilar to those from mice given mRNA vaccine where at alltested doses, the PCA displayed substantial overlap with placebo (mRNA:shown as center sphere in FIG. 2D, large upright sphere in FIG. 2E, andas flat line with four data points along the bottom of the center squarein FIG. 2F; placebo (PBS control): shown as upper sphere in FIG. 2D,lower elongated and narrow sphere in FIG. 2E, and upper sphere in FIG.2F).

Differentially expressed genes in the lymph nodes of mice given STARR™SARS-CoV-2 RNA compared to those inoculated with mRNA vaccine wereassessed next. Volcano plot analysis identified significant upregulationof several innate, B cell, and T cells genes in STARR™ SARS-CoV-2 RNAimmunized animals (FIG. 2G-2I). Some of the most highly differentiallyexpressed genes included, for example, GZMB (important for target cellkilling by cytotoxic immune cells), S100A8 and S100A9 (factors thatregulate immune responses via TLR4), TNFRSF17 (also known as BCMA andregulates humoral immunity), CXCR3 (chemokine receptor involved in Tcell trafficking and function) and AICDA (mediates antibody classswitching and somatic hypermutation in B cells).

These findings collectively indicate that the adaptive immune responsesin the draining lymph nodes of mice inoculated with STARR™ SARS-CoV-2RNA appeared to be significantly different compared to immune responsesin mice inoculated with a non-replicating mRNA vaccine.

Example 3

This example describes STARR™ SARS-CoV-2 RNA-induced T cell responses.

The cellular immune response following vaccination of C57BL/6 mice (n=5per group) with mRNA or the STARR™ SARS-CoV-2 RNA construct encoding theSARS-CoV-2 glycoprotein described above (Example 1) was investigatednext. At day 7 post-vaccination, spleens were harvested and assessed forCD8 and CD4 T cells by flow-cytometry. The CD8+ T cell CD44+CD62L−effector/memory subset was significantly expanded in STARR™ SARS-CoV-2RNA vaccinated mice compared to those given either PBS or mRNA vaccine(FIG. 3A-B). There was no statistically significant difference in theproportion of CD4+ T effector cells of these animals (FIG. 3C).IFNγ+CD8+ T cells (with 2 μg and 10 μg doses) and IFNγ+CD4+ T cells (in0.2 μg and 10 μg) were proportionately higher, as found usingintracellular staining (ICS) with flow cytometry, in STARR™ SARS-CoV-2RNA as compared to mRNA vaccinated animals (FIG. 3D-3F).

SARS-CoV-2 specific cellular responses were assessed in vaccinatedanimals by ELISPOT. A set of 15-mer peptides covering the SARS-CoV-2 Sprotein were divided into 4 pools and tested for IFNγ+ responses insplenocytes of vaccinated and non-vaccinated animals.SARS-CoV-2-specific cellular responses (displayed as IFNγ+ SFU/106cells) were detected by ELISPOT in both STARR™ SARS-CoV-2 RNA and mRNAvaccine immunized animals compared to PBS control (FIG. 3G-3I). Theseresponses were higher across the doses in STARR™ SARS-CoV-2 RNA comparedto mRNA vaccinated groups (FIG. 3G-3I). Even the highest tested dose (10μg) of mRNA vaccine produced IFNγ+ ELISPOT responses that wereappreciably lower than those by the lowest dose (0.2 μg) of STARR™SARS-CoV-2 RNA.

These results show that the STARR™ SARS-CoV-2 RNA construct inducedstrong T cell responses.

Example 4

This example illustrates humoral responses following vaccination withSTARR™ SARS-CoV-2 RNA.

SARS-CoV-2-specific humoral responses following vaccination werecharacterized in two different mouse models, BALB/c and C57BL/6. Femalemice (n=5 per group) were vaccinated at day 0 and bled every 10 days, upto day 60 for BALB/c and day 30 for C57BL/6 (FIG. 4A). SARS-CoV-2S-specific IgM responses were tested at 1:2000 serum dilution using anin-house Luminex immuno-assay. All tested doses of mRNA vaccine andSTARR™ SARS-CoV-2 RNA (corresponding to SEQ ID NO:125, as described inExample 1 above) produced detectable S-specific IgM responses in bothmouse models (FIG. 4B-4C). When comparing mRNA to STARR™ SARS-CoV-2 RNAvaccinated BALC/c mice, no difference in IgM responses was observed; IgMlevels in C57BL/6 mice were higher in STARR™ SARS-CoV-2 RNA vaccinatedC57BL/6 mice at day 10 post vaccination. In contrast, SARS-CoV-2S-specific IgG (at 1:2000 serum dilution) levels were higher from day 20onwards in animals inoculated with STARR™ SARS-CoV-2 RNA compared tomRNA vaccine (FIG. 4D-4E). Remarkably, the IgG levels continued to showan increasing trend in STARR™ SARS-CoV-2 RNA vaccinated mice, bothBALB/c and C57BL/6, until day 50 post-vaccination with a singleinoculation across all the doses. This trend contrasted with mice thatreceived the mRNA vaccine where in BALB/c mice antibody levels plateauedafter day 10 post-vaccination; increasing S-specific IgG levels wereobserved in mRNA-vaccinated C57BL/6 mice but these were lower than thoseseen in mice that received STARR™ SARS-CoV-2 RNA.

Further characterization of the SARS-CoV-2 specific IgG response invaccinated animals was conducted at day 30 post-immunization to assesswhich regions of the S protein are targeted. IgG endpoint titers wereestimated to full ectodomain S protein, S1, S2 and receptor bindingdomain (RBD) regions. For both vaccine candidates the majority ofSARS-CoV-2 specific IgG recognized S1, although high IgG endpoint titerswere also detected to S2 protein (FIG. 4F-4G). However, STARR™SARS-CoV-2 RNA elicited IgG endpoint titers were significantly highercompared to those produced by mRNA vaccination (FIG. 4F-4G). Notably,IgG that bind the receptor binding domain (RBD) of S protein, which isan immunodominant site of neutralizing antibodies, were also higher inSTARR™ SARS-CoV-2 RNA compared to mRNA vaccinated animals. Furthermore,at lower doses, mRNA vaccine but not STARR™ SARS-CoV-2 RNA struggled toelicit high SARS-CoV-2 specific IgG titers in the more Th1 dominantC57BL/6 mouse strain (FIG. 4G). Taken collectively, a single dose ofSTARR™ SARS-CoV-2 RNA induced significant differences in immune geneexpression and superior cellular immune responses in draining lymphnodes compared to mRNA vaccine and consequently humoral immuneresponses.

These data show that STARR™ SARS-CoV-2 RNA vaccination induced elevatedhumoral responses as compared to mRNA vaccination.

Example 5

This example illustrates reduced risk of immune enhancement upon STARR™SARS-CoV-2 RNA vaccination.

A safety consideration for coronavirus vaccine is a risk ofvaccine-mediate immune enhancement of respiratory disease. Indeed,SARS-CoV and MERS-CoV vaccine development have highlighted theimportance of Th1 skewed responses to avoid vaccine-induced immuneenhancement. Therefore, the Th1/Th2 balance elicited by both mRNA andSTARR™ SARS-CoV-2 RNA (self-replicating RNA construct as described inExample 1 above) vaccination was investigated. The IgG subclass fate ofplasma cells is influenced by T helper (Th) cells. At day 30post-vaccination, both mRNA and STARR™ SARS-CoV-2 RNA, except the 0.2 μgdose in C56BL/6J mice, induced comparable amounts of SARS-CoV-2S-specific IgG1, a Th2-associated IgG subclass in mice (FIG. 5A-5B). Incontrast, the Th1-associated IgG subclasses—IgG2a in BALB/c and IgG2c inC56BL/6J—were significantly greater in STARR™ SARS-CoV-2 RNA vaccinatedanimals. The ratios of S protein-specific IgG2a/IgG1 (BALBc) andIgG2c/IgG1 (C57BL/6) were greater than 1 in STARR™ SARS-CoV-2 RNAvaccinated animals (FIG. 5A-5B). Except for the 0.2 ug dose, theseratios were all significantly greater with STARR™ SARS-CoV-2 RNAcompared to mRNA vaccinated animals.

ICS was used to investigate the production of IFNγ (Th1 cytokine) andIL4 (Th2 cytokine) by CD4+ T cells in spleens of day 7 vaccinatedC56BL/6J mice. As shown above (Example 3), compared to mRNA vaccination,IFNγ levels were significantly greater in STARR™ SARS-CoV-2 RNAvaccinated animals (FIG. 3F). IL4 expression in CD4 T cells was slightlyhigher in mRNA as compared to STARR™ SARS-CoV-2 RNA at 0.2 μg and 2 μgdoses (FIG. 5C). In comparing the IFNγ and IL4 levels in individualmice, the ratios of IFNγ/IL4 in CD4+ T cells for both STARR™ SARS-CoV-2RNA and mRNA vaccinated mice were above 1 (FIG. 5D). The ratio ofIFNγ/IL4 in CD4+ T cells in mice given the 0.2 μg and 2 μg doses weresignificantly greater with STARR™ SARS-CoV-2 RNA than mRNA vaccination(FIG. 5F). However, without being limited by theory, the elevated ratiosin these doses appeared to be due to the lowered IL4 expression atlevels below background (i.e., PBS control mice), rather than reducedIFNγ and hence Th1 activity.

Taken collectively, these data show that STARR™ SARS-CoV-2 RNA producedTh1 instead of Th2 skewed adaptive immune responses.

Example 6

This example illustrates the quality of STARR™ SARS-CoV-2 RNA-inducedhumoral immune responses.

The binding strength (avidity) and the neutralizing ability of theantibody response elicited by the self-replicating STARR™ SARS-CoV-2 RNA(construct as described in Example 1 above) and mRNA vaccine constructswas assessed next. Serum IgG avidity was measured at day 30post-vaccination using a modified Luminex immuno-assay with 8M ureawashes. STARR™ SARS-CoV-2 RNA elicited higher avidity S protein-specificIgG than mRNA in both mouse models at all tested doses (FIG. 6A). Thesedifferences were observed, with the exception of 0.2 μg in BALB/c,across all doses (FIG. 6A), indicating that STARR™ SARS-CoV-2 RNAelicited better quality antibodies than conventional mRNA.

Neutralization of live SARS-CoV-2 by serum from vaccinated animals wasassessed using the plaque reduction neutralization test (PRNT). At day30 STARR™ SARS-CoV-2 RNA vaccinated BALB/c showed a clear dose dependentelevation in PRNT50 titers; 4 out of 5 (80%) of mice in the 10 μg STARR™SARS-CoV-2 RNA group showed PRNT50 titers above the 320 upper limit(FIG. 6B). Similar dose-dependent trends in PRNT50 titers were alsofound in C57B/6 mice, although in these animals, the PRNT50 titers ofseveral animals exceeded the 320 upper limit even with a low 0.2 μg dosevaccination (FIG. 6B). In contrast, PRNT50 titers in animals inoculatedwith mRNA vaccine construct were, except for one C57BL/6J mouse thatreceived 10 μg dose, all <20 (FIG. 6B). Unexpectedly and surprisingly,PRNT50 and PRNT70 titers of STARR™ SARS-CoV-2 RNA vaccinated BALBc micecontinued to rise between day 30 and day 60 after a single dose ofvaccination (FIG. 6C-6D). These titers were also comparable to PRNT70titers in sera from convalescent COVID-19 patients (FIG. 6D).

S protein IgG titers also positively correlated with PRNT50 titers inboth mouse models (FIG. 6E). Similar positive correlations were alsoobserved with IgG against S1 and RBD (FIG. 9 ). By contrast, nocorrelation was found between IgG and PRNT50 titers in mRNA vaccinatedmice (FIG. 6E). Taken collectively, without being limited by theory,these antibody response analyses indicate that the higher PRNT50 titersfollowing STARR™ SARS-CoV-2 RNA vaccination are not only attributable tothe amount of IgG produced but also due to superiority of the quality ofthe anti-SARS-CoV-2 antibodies.

In summary, STARR™ SARS-CoV-2 RNA induced qualitatively superior humoralimmune response than conventional mRNA.

Example 7

This example illustrates the effect of a second dose of STARR™SARS-CoV-2 RNA.

A possible added benefit of a second dose of STARR™ SARS-CoV-2 RNA(self-replicating RNA construct as described in Example 1 above) to thecellular and humoral immune responses to the S protein of SARS-CoV-2 wasexplored next. The clinical scores after the second dose were higherthan after the first dose (FIG. 7A). Like the first dose, mice thatreceived 2 μg and 10 μg of STARR™ SARS-CoV-2 RNA experienced weight loss(FIG. 7B). The IgG response to a second dose of STARR™ SARS-CoV-2 RNAproduced an appreciable boost in S protein-specific IgG levels, but onlywith 0.2 μg and 2 μg of STARR™ SARS-CoV-2 RNA (FIG. 7C). Without beinglimited by theory, a likely reason for the lack of increase in theanti-S protein specific IgG levels for the 10 μg dose is that the amountof fluorescence is near the saturation point of the detector and serawas not further diluted to observe and increase. However, in asubsequent Balb/c mouse study, the sera from mice vaccinated with a 5 μgRNA dose administered unilaterally in a 0.05 mL injection volumeproduced a significant increase in neutralizing antibody titers asassayed using a 96 well microneutralization assay format. Mice were bledevery 14 days and a second vaccination of 5 μg was administered on day28. 4 mice were injected with a VEEV replicon RNA expressing luciferaseas a negative control and 6 mice were vaccinated with STARR™ SARS-CoV-2RNA. The results are shown in Table 8 below.

TABLE 7 Microneutralization Titers (MN50) in Balb/c Mice MouseMicroneutralization Titers (MN50) No. Treatment Wk 0 Wk2 Wk4 Wk6 Wk8 1Luciferase <10 <10 <10 <10 <10 2 Luciferase <10 <10 <10 <10 <10 3Luciferase <10 <10 <10 <10 <10 4 Luciferase <10 <10 <10 <10 <10 5STARR ™ SARS- <10 1,280 5,120 327,680 81,920 CoV-2 RNA 6 STARR ™ SARS-<10 640 20,480 327,680 327,680 CoV-2 RNA 7 STARR ™ SARS- <10 1,280 2,560163,840 163,840 CoV-2 RNA 8 STARR ™ SARS- <10 1,280 10,240 327,680163,840 CoV-2 RNA 9 STARR ™ SARS- <10 640 40,960 327,680 327,680 COV-2RNA 10  STARR ™ SARS- <10 1,280 10,240 327,680 327,680 CoV-2 RNA Avg1,016 10,240 29,1930 206,426 Geometric Mean

The neutralization titers increased ˜10 fold between day 14 and day 28post vaccination. Following the boost on day 28, the neutralizationtiters increased an additional 20 fold 14 days post boost.

To determine if there was added benefit in IFNγ+CD8+ T cell counts froma second dose vaccination, CD8 T cell IFNγ responses in mice given onlya prime were compared to responses of mice given a prime and a boost.Fold change in IFNγ+CD8+ T cells in the vaccinated over PBS control micewas calculated for mice given either a prime only or given a prime andboost. The fold change of IFNγ+CD8+ T cells was similar following theprime and prime+boost for 2 μg and 10 μg doses of STARR™ SARS-CoV-2 RNA(FIG. 7D-7E); the 0.2 μg dose showed higher fold change of IFNγ+CD8+ Tcells between prime (at day 7) and prime+boost (day 50). Vaccinationwith 0.2 μg of mRNA also showed increased IFNγ+CD8+ T cells relative toPBS control after two doses of vaccination. Without being limited bytheory, these findings suggest that a second 10 μg dose of STARR™SARS-CoV-2 RNA did not produce superior cellular immunity compared tosingle dose vaccination. Thus, there was no apparent benefit from asecond 10 μg dose of STARR™ SARS-CoV-2 RNA.

Taken collectively, these data suggest that 10 μg STARR™ SARS-CoV-2 RNAoffers the opportunity of a single dose vaccination to protect againstCOVID-19.

Example 8

This example illustrates protection from SARS-CoV-2 viral challenge inmice following vaccination with STARR™ SARS-CoV-2 self-replicating RNA.

A mouse viral challenge study was conducted with human ACE2 transgenicmice. Mice were immunized with 2 μg and 10 μg RNA doses of STARR™SARS-CoV-2 RNA (RNA construct as described in Example 1 above) orinjected with PBS. There were three different cohorts with 5 mice ineach treatment group. Cohorts 1 and 3 received a lethal SARS-CoV-2 viruschallenge load of 5×105 TCID50. Cohort 1 was monitored for survival andCohort 3 was euthanized 5 days after challenge. Lungs were assayed forviral load and processed for histopathology. Cohorts 2 received asublethal viral load of 5×104 TCID50. Cohort 2 was euthanized 5 daysafter virus challenge and lungs were assayed for infectious virus andprocessed for histopathology. All mice were inoculated intratracheally30 days post-vaccination with a single dose of STARR™ SARS-CoV-2 RNA.

All mice injected with PBS in cohort 1 were dead by day 7, whereas allvaccinated mice showed no signs of infection 15 days after viralchallenge (FIG. 10 ). For Cohort 2 receiving a sublethal viral load, 10to 3,300 copies of virus was measured by RT-PCR in the lungs with anaverage of 1,200 copies, whereas no copies of viral RNA were measured inmice vaccinated with ARTC-021 at 2 μg and 10 μg RNA doses (LOD was 0.1copies; FIG. 11 , left). Copies of viral RNA were also observed in thebrain ranging from 20 to 80 in the PBS treatment group, whereas no viralRNA copies were measured in the brains of mice vaccinated with either2.0 μg or 10.0 μg RNA doses (FIG. 11 , right). Lastly, lungs werecarefully processed and assayed for lung plaque titers. The averageplaque titers for the group injected with PBS was 8×103/mL of lunghomogenate, whereas no plaques were detected for mice vaccinated witheither 2.0 μg or 10.0 μg or STARR™ SARS-CoV-2 RNA (FIG. 12 ). Lung andbrain tissues from Cohort 3 are being assayed for viral copy number andinfectious virus. Histopathology of lungs for cohorts 2 and 3 is inprogress.

These results show that vaccination with STARR™ SARS-CoV-2self-replicating RNA protected mice from a lethal SARS-CoV-2 infectionand protected against lung and brain infection upon challenge with asublethal dose of SARS-CoV-2.

Example 9

The COVID-19 pandemic is caused by infection with the SARS-CoV-2 virus.A major mutation detected to date in the SARS-CoV-2 viral envelope spikeprotein, which is responsible for virus attachment to the host and isalso the main target for host antibodies, is a mutation of an aspartate(D) at position 614 found frequently in Chinese strains to a glycine(G). VEEV Replicon transcripts expressing the D614 and G614 versions ofthe SARS-CoV-2 spike glycoprotein were formulated with the exact samelipid formulation as studies described in Examples 1-8. Balb/c mice werevaccinated with a single RNA administration of 0.2 μg, 2.0 μg and 10.0μg of RNA. There were 5 mice per dose. Mice were bled on days, 14, 28and 42 post vaccination. Sera was diluted 1/2000 and incubated withLuminex beads derivatized with the SARS-CoV-2 spike glycoproteincontaining the D614 amino acid sequence. A secondary mouse antibodyderivatized with a fluorophore was used to assay for bound antibody tothe beads and adjusted mean fluorescence intensity (MFI) was measured asa function of RNA dose, shown in FIG. 13 . The results showed that MFIincreased as a function of RNA dose with slightly higher MFI observedfor the serum from mice immunized with the G614 spike glycoprotein. Thisslight elevation is attributed to a lower percentage of full length RNAwith the D614 amino acid sequence. An important conclusion is that theserum from mice immunized with the G614 spike glycoprotein RNA constructwas able to bind to spike glycoprotein with the D614 amino acidsequence, indicative of cross reactivity.

These results show that immunization with a G614 spike glycoproteinexpressed from self-replicating RNA results in production of antibodiesthat are able to bind to a D614 spike glycoprotein.

Discussion of Examples 1-9

The pandemic of COVID-19 has necessitated rapid development of vaccines,as physical distancing to prevent SARS-CoV-2 transmission is not asustainable long-term solution. Several COVID-19 vaccine candidates arenow in clinical trials and more are entering first-in-human trials.However, a majority of vaccine candidates being developed require twodoses for sufficient adaptive immunity. A single dose vaccine thatgenerates both cellular and humoral immunity, without elevating the riskof vaccine-mediated immune enhancement, remains an unmet need. Withoutbeing limited by theory, deployment of a single dose vaccine wouldenable greater level of compliance and enable distribution of finiteproduction of vaccines to more susceptible people globally.

Among licensed vaccines, live attenuated vaccines can offer durableprotection against viral diseases. Live vaccines infect and replicate atsites of inoculation and some even in draining lymph nodes. Replicationenables endogenous expression of viral antigens that enables antigenpresentation to stimulate cytotoxic CD8+ T cells. Expressed antigenswould also be taken up by antigen presenting cells to trigger CD4+ Tcell help that drive affinity maturation in B cells. Studies on the liveattenuated yellow fever vaccine have shown that a longer period ofstimulation of the adaptive immune response results in superior adaptiveimmune responses. Without being limited by theory, simulating theprocesses of live vaccination could offer a chance of durable immunityagainst COVID-19.

In a crisis such as COVID-19, a nucleic acid vaccine platform offersopportunities for accelerated development. In studies described herein,a side-by-side comparison of the immunogenicity elicited by twoSARS-CoV-2 vaccines candidates was conducted, a non-replicative mRNAconstruct and STARR™ SARS-CoV-2 RNA. Compared to an mRNA vaccine, STARR™SARS-CoV-2 RNA produced higher and longer protein expression in vivo andupregulated gene expression of several innate, B cell, and T cellresponse genes in the blood and draining lymph nodes. These propertiestranslated into significantly greater CD8+ T cell responses, IFNγ+ELISPOT responses, and SARS-CoV-2 specific IgG and Th1 skewed responses.Interestingly, despite the highest tested dose of mRNA elicitingcomparable S protein-specific antibodies as the lowest tested dose ofSTARR™ SARS-CoV-2 RNA, mRNA-elicited IgG did not show similar avidity orneutralization activity as those from STARR™ SARS-CoV-2 RNA vaccination.These findings thus highlight the immunological advantages ofself-replicating RNA over mRNA platforms. In addition, mouse challengestudies with SARS-CoV-2 virus showed that vaccination with a single highdose (10 μg) or a single low dose (2 μg) of STARR™ SARS-CoV-2self-replicating RNA protected mice from a lethal SARS-CoV-2 infectionand protected from lung and brain infection upon challenge with asublethal SARS-CoV-2 dose.

The extent to which STARR™ vaccines reproduce the features of livevaccines remain to be experimentally defined. Without being limited bytheory, the superior quality of immune responses elicited by STARR™SARS-CoV-2 RNA over the mRNA vaccine construct could be attributable tomultiple factors, all of which have been found to be associated withlive vaccination. For example, higher and longer expression ofimmunogens produce better immunity, likely through better engagement ofT follicular helper cells and thereby leading to more diverse antibodytargets and more neutralizing antibody responses. Replication of STARR™SARS-CoV-2 RNA would result in the formation of a negative-strandtemplate for production of more positive-strand mRNA and sub-genomicmRNA expressing the S transgene. Interaction between the negative- andpositive-strands would form double stranded RNA (dsRNA), which wouldinteract with TLR3 and RIG-I-like receptors to stimulate interferonresponses, which has been shown to correlate with superior adaptiveimmune responses. Production of IFNγ can then stimulate development ofcytotoxic CD8+ T cells. Importantly, the S protein does contain humanCD8+ T cell epitopes. Without being limited by theory, the developmentof T cell memory could be important for long-term immunity, as suggestedby recent findings on T cell responses to SARS-CoV-2 and othercoronavirus infections.

It is unclear whether the VEEV nsP1-4 forming the replication complexcontains any immunogenic properties, although mutations in the nsPproteins have been shown to affect induction of type I IFN. VEEVreplicons have also been shown to adjuvant immune responses at mucosalsites, further illustrating the advantages of using the STARR™ platformto develop a COVID-19 vaccine. Without being limited by theory, theredoes not appear to be an immune response to replicon non-structuralproteins, as indicated by an increase in antigen-specific IgG productionupon a second administration of replicon to animals. In the presence ofan immune response to non-structural proteins, a limited or no increasein antigen-specific IgG production may have resulted following a secondadministration. The RNA is encapsulated in lipid nanoparticles (LNP),which together can form potent adjuvants leading to robust immuneresponses. In addition, using the genetic sequence of an antigen,including a viral antigen such as the spike protein from SARS-CoV-2, forexample, STARR™ vaccines can be rapidly generated and manufactured usingcell-free and rapidly scalable techniques.

In conclusion, a STARR™ vaccine as exemplified by STARR™ SARS-CoV-2 RNAoffers an approach to simulate several of the properties of livevaccination and offers a potential for single-dose vaccination againstCOVID-19.

SEQUENCES SEQ ID NO: 72ATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCCGTCGACGGCCCCACCAGCCTGTACCACCAGGCCAACAAGGGCGTGAGGGTGGCCTACTGGATCGGCTTCGACACCACACCCTTCATGTTCAAGAACCTGGCCGGCGCCTACCCCAGCTACAGCACCAACTGGGCCGACGAGACCGTGCTGACCGCCAGGAACATCGGCCTGTGCAGCAGCGACGTGATGGAGAGGAGCCGGAGAGGCATGAGCATCCTGAGGAAGAAATACCTGAAGCCCAGCAACAACGTGCTGTTCAGCGTGGGCAGCACCATCTACCACGAGAAGAGGGACCTGCTCAGGAGCTGGCACCTGCCCAGCGTGTTCCACCTGAGGGGCAAGCAGAACTACACCTGCAGGTGCGAGACCATCGTGAGCTGCGACGGCTACGTGGTGAAGAGGATCGCCATCAGCCCCGGCCTGTACGGCAAGCCCAGCGGCTACGCCGCTACAATGCACAGGGAGGGCTTCCTGTGCTGCAAGGTGACCGACACCCTGAACGGCGAGAGGGTGAGCTTCCCCGTGTGCACCTACGTGCCCGCCACCCTGTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGAGCGCCGACGACGCCCAGAAGCTGCTCGTGGGCCTGAACCAGAGGATCGTGGTCAACGGCAGGACCCAGAGGAACACCAACACAATGAAGAACTACCTGCTGCCCGTGGTGGCCCAGGCTTTCGCCAGGTGGGCCAAGGAGTACAAGGAGGACCAGGAAGACGAGAGGCCCCTGGGCCTGAGGGACAGGCAGCTGGTGATGGGCTGCTGCTGGGCCTTCAGGCGGCACAAGATCACCAGCATCTACAAGAGGCCCGACACCCAGACCATCATCAAGGTGAACAGCGACTTCCACAGCTTCGTGCTGCCCAGGATCGGCAGCAACACCCTGGAGATCGGCCTGAGGACCCGGATCAGGAAGATGCTGGAGGAACACAAGGAGCCCAGCCCACTGATCACCGCCGAGGACGTGCAGGAGGCCAAGTGCGCTGCCGACGAGGCCAAGGAGGTGAGGGAGGCCGAGGAACTGAGGGCCGCCCTGCCACCCCTGGCTGCCGACGTGGAGGAACCCACCCTGGAAGCCGACGTGGACCTGATGCTGCAGGAGGCCGGCGCCGGAAGCGTGGAGACACCCAGGGGCCTGATCAAGGTGACCAGCTACGACGGCGAGGACAAGATCGGCAGCTACGCCGTGCTGAGCCCACAGGCCGTGCTGAAGTCCGAGAAGCTGAGCTGCATCCACCCACTGGCCGAGCAGGTGATCGTGATCACCCACAGCGGCAGGAAGGGCAGGTACGCCGTGGAGCCCTACCACGGCAAGGTGGTCGTGCCCGAGGGCCACGCCATCCCCGTGCAGGACTTCCAGGCCCTGAGCGAGAGCGCCACCATCGTGTACAACGAGAGGGAGTTCGTGAACAGGTACCTGCACCATATCGCCACCCACGGCGGAGCCCTGAACACCGACGAGGAATACTACAAGACCGTGAAGCCCAGCGAGCACGACGGCGAGTACCTGTACGACATCGACAGGAAGCAGTGCGTGAAGAAAGAGCTGGTGACCGGCCTGGGACTGACCGGCGAGCTGGTGGACCCACCCTTCCACGAGTTCGCCTACGAGAGCCTGAGGACCAGACCCGCCGCTCCCTACCAGGTGCCCACCATCGGCGTGTACGGCGTGCCCGGCAGCGGAAAGAGCGGCATCATCAAGAGCGCCGTGACCAAGAAAGACCTGGTGGTCAGCGCCAAGAAAGAGAACTGCGCCGAGATCATCAGGGACGTGAAGAAGATGAAAGGCCTGGACGTGAACGCGCGCACCGTGGACAGCGTGCTGCTGAACGGCTGCAAGCACCCCGTGGAGACCCTGTACATCGACGAGGCCTTCGCTTGCCACGCCGGCACCCTGAGGGCCCTGATCGCCATCATCAGGCCCAAGAAAGCCGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATGATGTGCCTGAAGGTGCACTTCAACCACGAGATCTGCACCCAGGTGTTCCACAAGAGCATCAGCAGGCGGTGCACCAAGAGCGTGACCAGCGTCGTGAGCACCCTGTTCTACGACAAGAAAATGAGGACCACCAACCCCAAGGAGACCAAAATCGTGATCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCTGATCCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCAGATCGACTACAAGGGCAACGAGATCATGACCGCCGCTGCCAGCCAGGGCCTGACCAGGAAGGGCGTGTACGCCGTGAGGTACAAGGTGAACGAGAACCCACTGTACGCTCCCACCAGCGAGCACGTGAACGTGCTGCTGACCAGGACCGAGGACAGGATCGTGTGGAAGACCCTGGCCGGCGACCCCTGGATCAAGACCCTGACCGCCAAGTACCCCGGCAACTTCACCGCCACCATCGAAGAGTGGCAGGCCGAGCACGACGCCATCATGAGGCACATCCTGGAGAGGCCCGACCCCACCGACGTGTTCCAGAACAAGGCCAACGTGTGCTGGGCCAAGGCCCTGGTGCCCGTGCTGAAGACCGCCGGCATCGACATGACCACAGAGCAGTGGAACACCGTGGACTACTTCGAGACCGACAAGGCCCACAGCGCCGAGATCGTGCTGAACCAGCTGTGCGTGAGGTTCTTCGGCCTGGACCTGGACAGCGGCCTGTTCAGCGCCCCCACCGTGCCACTGAGCATCAGGAACAACCACTGGGACAACAGCCCCAGCCCAAACATGTACGGCCTGAACAAGGAGGTGGTCAGGCAGCTGAGCAGGCGGTACCCACAGCTGCCCAGGGCCGTGGCCACCGGCAGGGTGTACGACATGAACACCGGCACCCTGAGGAACTACGACCCCAGGATCAACCTGGTGCCCGTGAACAGGCGGCTGCCCCACGCCCTGGTGCTGCACCACAACGAGCACCCACAGAGCGACTTCAGCTCCTTCGTGAGCAAGCTGAAAGGCAGGACCGTGCTGGTCGTGGGCGAGAAGCTGAGCGTGCCCGGCAAGATGGTGGACTGGCTGAGCGACAGGCCCGAGGCCACCTTCCGGGCCAGGCTGGACCTCGGCATCCCCGGCGACGTGCCCAAGTACGACATCATCTTCGTGAACGTCAGGACCCCATACAAGTACCACCATTACCAGCAGTGCGAGGACCACGCCATCAAGCTGAGCATGCTGACCAAGAAGGCCTGCCTGCACCTGAACCCCGGAGGCACCTGCGTGAGCATCGGCTACGGCTACGCCGACAGGGCCAGCGAGAGCATCATTGGCGCCATCGCCAGGCTGTTCAAGTTCAGCAGGGTGTGCAAACCCAAGAGCAGCCTGGAGGAAACCGAGGTGCTGTTCGTGTTCATCGGCTACGACCGGAAGGCCAGGACCCACAACCCCTACAAGCTGAGCAGCACCCTGACAAACATCTACACCGGCAGCAGGCTGCACGAGGCCGGCTGCGCCCCCAGCTACCACGTGGTCAGGGGCGATATCGCCACCGCCACCGAGGGCGTGATCATCAACGCTGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGTGTGCGGCGCCCTGTACAAGAAGTTCCCCGAGAGCTTCGACCTGCAGCCCATCGAGGTGGGCAAGGCCAGGCTGGTGAAGGGCGCCGCTAAGCACATCATCCACGCCGTGGGCCCCAACTTCAACAAGGTGAGCGAGGTGGAAGGCGACAAGCAGCTGGCCGAAGCCTACGAGAGCATCGCCAAGATCGTGAACGACAATAACTACAAGAGCGTGGCCATCCCACTGCTCAGCACCGGCATCTTCAGCGGCAACAAGGACAGGCTGACCCAGAGCCTGAACCACCTGCTCACCGCCCTGGACACCACCGATGCCGACGTGGCCATCTACTGCAGGGACAAGAAGTGGGAGATGACCCTGAAGGAGGCCGTGGCCAGGCGGGAGGCCGTGGAAGAGATCTGCATCAGCGACGACTCCAGCGTGACCGAGCCCGACGCCGAGCTGGTGAGGGTGCACCCCAAGAGCTCCCTGGCCGGCAGGAAGGGCTACAGCACCAGCGACGGCAAGACCTTCAGCTACCTGGAGGGCACCAAGTTCCACCAGGCCGCTAAGGACATCGCCGAGATCAACGCTATGTGGCCCGTGGCCACCGAGGCCAACGAGCAGGTGTGCATGTACATCCTGGGCGAGAGCATGTCCAGCATCAGGAGCAAGTGCCCCGTGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCTGCCCTGCCTGTGCATCCACGCTATGACACCCGAGAGGGTGCAGCGGCTGAAGGCCAGCAGGCCCGAGCAGATCACCGTGTGCAGCTCCTTCCCACTGCCCAAGTACAGGATCACCGGCGTGCAGAAGATCCAGTGCAGCCAGCCCATCCTGTTCAGCCCAAAGGTGCCCGCCTACATCCACCCCAGGAAGTACCTGGTGGAGACCCCACCCGTGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCTGATCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCATCATTATCGAGGAAGAGGAAGAGGACAGCATCAGCCTGCTGAGCGACGGCCCCACCCACCAGGTGCTGCAGGTGGAGGCCGACATCCACGGCCCACCCAGCGTGTCCAGCTCCAGCTGGAGCATCCCACACGCCAGCGACTTCGACGTGGACAGCCTGAGCATCCTGGACACCCTGGAGGGCGCCAGCGTGACCTCCGGCGCCACCAGCGCCGAGACCAACAGCTACTTCGCCAAGAGCATGGAGTTCCTGGCCAGGCCCGTGCCAGCTCCCAGGACCGTGTTCAGGAACCCACCCCACCCAGCTCCCAGGACCAGGACCCCAAGCCTGGCTCCCAGCAGGGCCTGCAGCAGGACCAGCCTGGTGAGCACCCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAACTGGAGGCCCTGACACCCAGCAGGACCCCCAGCAGGTCCGTGAGCAGGACTAGTCTGGTGTCCAACCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAATTCGAGGCCTTCGTGGCCCAGCAACAGAGACGGTTCGACGCCGGCGCCTACATCTTCAGCAGCGACACCGGCCAGGGACACCTGCAGCAAAAGAGCGTGAGGCAGACCGTGCTGAGCGAGGTGGTGCTGGAGAGGACCGAGCTGGAAATCAGCTACGCCCCCAGGCTGGACCAGGAGAAGGAGGAACTGCTCAGGAAGAAACTGCAGCTGAACCCCACCCCAGCCAACAGGAGCAGGTACCAGAGCAGGAAGGTGGAGAACATGAAGGCCATCACCGCCAGGCGGATCCTGCAGGGCCTGGGACACTACCTGAAGGCCGAGGGCAAGGTGGAGTGCTACAGGACCCTGCACCCCGTGCCACTGTACAGCTCCAGCGTGAACAGGGCCTTCTCCAGCCCCAAGGTGGCCGTGGAGGCCTGCAACGCTATGCTGAAGGAGAACTTCCCCACCGTGGCCAGCTACTGCATCATCCCCGAGTACGACGCCTACCTGGACATGGTGGACGGCGCCAGCTGCTGCCTGGACACCGCCAGCTTCTGCCCCGCCAAGCTGAGGAGCTTCCCCAAGAAACACAGCTACCTGGAGCCCACCATCAGGAGCGCCGTGCCCAGCGCCATCCAGAACACCCTGCAGAACGTGCTGGCCGCTGCCACCAAGAGGAACTGCAACGTGACCCAGATGAGGGAGCTGCCCGTGCTGGACAGCGCTGCCTTCAACGTGGAGTGCTTCAAGAAATACGCCTGCAACAACGAGTACTGGGAGACCTTCAAGGAGAACCCCATCAGGCTGACCGAAGAGAACGTGGTGAACTACATCACCAAGCTGAAGGGCCCCAAGGCCGCTGCCCTGTTCGCTAAGACCCACAACCTGAACATGCTGCAGGACATCCCAATGGACAGGTTCGTGATGGACCTGAAGAGGGACGTGAAGGTGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGTGCAGGTGATCCAGGCCGCTGACCCACTGGCCACCGCCTACCTGTGCGGCATCCACAGGGAGCTGGTGAGGCGGCTGAACGCCGTGCTGCTGCCCAACATCCACACCCTGTTCGACATGAGCGCCGAGGACTTCGACGCCATCATCGCCGAGCACTTCCAGCCCGGCGACTGCGTGCTGGAGACCGACATCGCCAGCTTCGACAAGAGCGAGGATGACGCTATGGCCCTGACCGCTCTGATGATCCTGGAGGACCTGGGCGTGGACGCCGAGCTGCTCACCCTGATCGAGGCTGCCTTCGGCGAGATCAGCTCCATCCACCTGCCCACCAAGACCAAGTTCAAGTTCGGCGCTATGATGAAAAGCGGAATGTTCCTGACCCTGTTCGTGAACACCGTGATCAACATTGTGATCGCCAGCAGGGTGCTGCGGGAGAGGCTGACCGGCAGCCCCTGCGCTGCCTTCATCGGCGACGACAACATCGTGAAGGGCGTGAAAAGCGACAAGCTGATGGCCGACAGGTGCGCCACCTGGCTGAACATGGAGGTGAAGATCATCGACGCCGTGGTGGGCGAGAAGGCCCCCTACTTCTGCGGCGGATTCATCCTGTGCGACAGCGTGACCGGCACCGCCTGCAGGGTGGCCGACCCCCTGAAGAGGCTGTTCAAGCTGGGCAAGCCACTGGCCGCTGACGATGAGCACGACGATGACAGGCGGAGGGCCCTGCACGAGGAAAGCACCAGGTGGAACAGGGTGGGCATCCTGAGCGAGCTGTGCAAGGCCGTGGAGAGCAGGTACGAGACCGTGGGCACCAGCATCATCGTGATGGCTATGACCACACTGGCCAGCTCCGTCAAGAGCTTCTCCTACCTGAGGGGGGCCCCTATAACTCTCTACGGCTAA

SEQ ID NO: 73 ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA

SEQ ID NO: 74 GATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA

SEQ ID NO: 75 GATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA

SEQ ID NO: 76 ACTCGAGTATGTTACGTGCAAAGGTGATTGTCACCCCCCGAAAGACCATATTGTGACACACCCTCAGTATCACGCCCAAACATTTACAGCCGCGGTGTCAAAAACCGCGTGGACGTGGTTAACATCCCTGCTGGGAGGATCAGCCGTAATTATTATAATTGGCTTGGTGCTGGCTACTATTGTGGCCATGTACGTGCTGACCAACCAGAAACATAATTGAATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAATCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 121 ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTICAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTAC ATTACACATAA

SEQ ID NO: 122 ATGTTCGTCTTCCTGGTCCTGCTGCCTCTGGTCTCCTCACAGTGCGTCAATCTGACAACTCGGACTCAGCTGCCACCTGCTTATACTAATAGCTTCACCAGAGGCGTGTACTATCCTGACAAGGTGTTTAGAAGCTCCGTGCTGCACTCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAATGGCACAAAGCGGTTCGACAATCCCGTGCTGCCTTTTAACGATGGCGTGTACTTCGCCTCTACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTTGGCACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGTCATCAAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAATAAGAGCTGGATGGAGTCCGAGTTTAGAGTGTATTCTAGCGCCAACAACTGCACATTTGAGTACGTGAGCCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGACGGCTACTTCAAAATCTACTCTAAGCACACCCCCATCAACCTGGTGCGCGACCTGCCTCAGGGCTTCAGCGCCCTGGAGCCCCTGGTGGATCTGCCTATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAGCGGATGGACCGCCGGCGCTGCCGCCTACTATGTGGGCTACCTCCAGCCCCGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATCACAGACGCAGTGGATTGCGCCCTGGACCCCCTGAGCGAGACAAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATCAGACATCCAATTTCAGGGTGCAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGTTCAACGCAACCCGCTTCGCCAGCGTGTACGCCTGGAATAGGAAGCGGATCAGCAACTGCGTGGCCGACTATAGCGTGCTGTACAACTCCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACCTGTGCTTTACCAACGTCTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGCCCCCGGCCAGACAGGCAAGATCGCAGACTACAATTATAAGCTGCCAGACGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGGCAACTACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCTACAGAAATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCCACTCCAGTCCTACGGCTTCCAGCCCACAAACGGCGTGGGCTATCAGCCTTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCCCCAGCAACAGTGTGCGGCCCCAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGCACAGGCGTGCTGACCGAGTCCAACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGACCCACAGACCCTGGAGATCCTGGACATCACACCCTGCTCTTTCGGCGGCGTGAGCGTGATCACACCCGGCACCAATACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGTACCGAGGTGCCCGTGGCTATCCACGCCGATCAGCTGACCCCAACATGGCGGGTGTACAGCACCGGCTCCAACGTCTTCCAGACAAGAGCCGGATGCCTGATCGGAGCAGAGCACGTGAACAATTCCTATGAGTGCGACATCCCAATCGGCGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCCAGACGGGCCCGGAGCGTGGCCTCCCAGTCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAACAGCGTGGCCTACTCTAACAATAGCATCGCCATCCCAACCAACTTCACAATCTCTGTGACCACAGAGATCCTGCCCGTGTCCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTACCGAGTGCAGCAACCTGCTGCTCCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCCCTGACAGGCATCGCCGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAAATCTACAAGACCCCCCCTATCAAGGACTTTGGCGGCTTCAATTTTTCCCAGATCCTGCCTGATCCATCCAAGCCTTCTAAGCGGAGCTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCAGCCAGGGACCTGATCTGCGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCACCCCTGCTGACAGATGAGATGATCGCACAGTACACAAGCGCCCTGCTGGCCGGCACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTCCAGATCCCCTTTGCCATGCAGATGGCCTATAGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCTGTCCTCTACAGCCAGCGCCCTGGGCAAGCTCCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCATCTCTAGCGTGCTGAATGACATCCTGAGCCGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACCGGCCGGCTCCAGAGCCTCCAGACCTATGTGACACAGCAGCTGATCAGGGCCGCCGAGATCAGGGCCAGCGCCAATCTGGCAGCAACCAAGATGTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGGGCTATCACCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTGGTGTTTCTGCACGTGACCTACGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAAGGCCCACTTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCTACGAGCCCCAGATCATCACCACAGACAACACCTTCGTGAGCGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCACTCCAGCCCGAGCTGGACAGCTTTAAGGAGGAGCTGGATAAGTATTTCAAGAATCACACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCTAAGAATCTGAACGAGAGCCTGATCGACCTCCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGACATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGACGAGGATGACTCTGAACCTGTGCTGAAGGGCGTGAAGCTGCATTACAC CTAA

SEQ ID NO: 123MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQUITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKG VKLHYT

SEQ ID NO: 77 CCTGAATGGACTACGACATAGTCTAGTCCGCCAAGGCCGCCACC

SEQ ID NO: 78 ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCCGTCGACGGCCCCACCAGCCTGTACCACCAGGCCAACAAGGGCGTGAGGGTGGCCTACTGGATCGGCTTCGACACCACACCCTTCATGTTCAAGAACCTGGCCGGCGCCTACCCCAGCTACAGCACCAACTGGGCCGACGAGACCGTGCTGACCGCCAGGAACATCGGCCTGTGCAGCAGCGACGTGATGGAGAGGAGCCGGAGAGGCATGAGCATCCTGAGGAAGAAATACCTGAAGCCCAGCAACAACGTGCTGTTCAGCGTGGGCAGCACCATCTACCACGAGAAGAGGGACCTGCTCAGGAGCTGGCACCTGCCCAGCGTGTTCCACCTGAGGGGCAAGCAGAACTACACCTGCAGGTGCGAGACCATCGTGAGCTGCGACGGCTACGTGGTGAAGAGGATCGCCATCAGCCCCGGCCTGTACGGCAAGCCCAGCGGCTACGCCGCTACAATGCACAGGGAGGGCTTCCTGTGCTGCAAGGTGACCGACACCCTGAACGGCGAGAGGGTGAGCTTCCCCGTGTGCACCTACGTGCCCGCCACCCTGTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGAGCGCCGACGACGCCCAGAAGCTGCTCGTGGGCCTGAACCAGAGGATCGTGGTCAACGGCAGGACCCAGAGGAACACCAACACAATGAAGAACTACCTGCTGCCCGTGGTGGCCCAGGCTTTCGCCAGGTGGGCCAAGGAGTACAAGGAGGACCAGGAAGACGAGAGGCCCCTGGGCCTGAGGGACAGGCAGCTGGTGATGGGCTGCTGCTGGGCCTTCAGGCGGCACAAGATCACCAGCATCTACAAGAGGCCCGACACCCAGACCATCATCAAGGTGAACAGCGACTTCCACAGCTTCGTGCTGCCCAGGATCGGCAGCAACACCCTGGAGATCGGCCTGAGGACCCGGATCAGGAAGATGCTGGAGGAACACAAGGAGCCCAGCCCACTGATCACCGCCGAGGACGTGCAGGAGGCCAAGTGCGCTGCCGACGAGGCCAAGGAGGTGAGGGAGGCCGAGGAACTGAGGGCCGCCCTGCCACCCCTGGCTGCCGACGTGGAGGAACCCACCCTGGAAGCCGACGTGGACCTGATGCTGCAGGAGGCCGGCGCCGGAAGCGTGGAGACACCCAGGGGCCTGATCAAGGTGACCAGCTACGACGGCGAGGACAAGATCGGCAGCTACGCCGTGCTGAGCCCACAGGCCGTGCTGAAGTCCGAGAAGCTGAGCTGCATCCACCCACTGGCCGAGCAGGTGATCGTGATCACCCACAGCGGCAGGAAGGGCAGGTACGCCGTGGAGCCCTACCACGGCAAGGTGGTCGTGCCCGAGGGCCACGCCATCCCCGTGCAGGACTTCCAGGCCCTGAGCGAGAGCGCCACCATCGTGTACAACGAGAGGGAGTTCGTGAACAGGTACCTGCACCATATCGCCACCCACGGCGGAGCCCTGAACACCGACGAGGAATACTACAAGACCGTGAAGCCCAGCGAGCACGACGGCGAGTACCTGTACGACATCGACAGGAAGCAGTGCGTGAAGAAAGAGCTGGTGACCGGCCTGGGACTGACCGGCGAGCTGGTGGACCCACCCTTCCACGAGTTCGCCTACGAGAGCCTGAGGACCAGACCCGCCGCTCCCTACCAGGTGCCCACCATCGGCGTGTACGGCGTGCCCGGCAGCGGAAAGAGCGGCATCATCAAGAGCGCCGTGACCAAGAAAGACCTGGTGGTCAGCGCCAAGAAAGAGAACTGCGCCGAGATCATCAGGGACGTGAAGAAGATGAAAGGCCTGGACGTGAACGCGCGCACCGTGGACAGCGTGCTGCTGAACGGCTGCAAGCACCCCGTGGAGACCCTGTACATCGACGAGGCCTTCGCTTGCCACGCCGGCACCCTGAGGGCCCTGATCGCCATCATCAGGCCCAAGAAAGCCGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATGATGTGCCTGAAGGTGCACTTCAACCACGAGATCTGCACCCAGGTGTTCCACAAGAGCATCAGCAGGCGGTGCACCAAGAGCGTGACCAGCGTCGTGAGCACCCTGTTCTACGACAAGAAAATGAGGACCACCAACCCCAAGGAGACCAAAATCGTGATCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCTGATCCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCAGATCGACTACAAGGGCAACGAGATCATGACCGCCGCTGCCAGCCAGGGCCTGACCAGGAAGGGCGTGTACGCCGTGAGGTACAAGGTGAACGAGAACCCACTGTACGCTCCCACCAGCGAGCACGTGAACGTGCTGCTGACCAGGACCGAGGACAGGATCGTGTGGAAGACCCTGGCCGGCGACCCCTGGATCAAGACCCTGACCGCCAAGTACCCCGGCAACTTCACCGCCACCATCGAAGAGTGGCAGGCCGAGCACGACGCCATCATGAGGCACATCCTGGAGAGGCCCGACCCCACCGACGTGTTCCAGAACAAGGCCAACGTGTGCTGGGCCAAGGCCCTGGTGCCCGTGCTGAAGACCGCCGGCATCGACATGACCACAGAGCAGTGGAACACCGTGGACTACTTCGAGACCGACAAGGCCCACAGCGCCGAGATCGTGCTGAACCAGCTGTGCGTGAGGTTCTTCGGCCTGGACCTGGACAGCGGCCTGTTCAGCGCCCCCACCGTGCCACTGAGCATCAGGAACAACCACTGGGACAACAGCCCCAGCCCAAACATGTACGGCCTGAACAAGGAGGTGGTCAGGCAGCTGAGCAGGCGGTACCCACAGCTGCCCAGGGCCGTGGCCACCGGCAGGGTGTACGACATGAACACCGGCACCCTGAGGAACTACGACCCCAGGATCAACCTGGTGCCCGTGAACAGGCGGCTGCCCCACGCCCTGGTGCTGCACCACAACGAGCACCCACAGAGCGACTTCAGCTCCTTCGTGAGCAAGCTGAAAGGCAGGACCGTGCTGGTCGTGGGCGAGAAGCTGAGCGTGCCCGGCAAGATGGTGGACTGGCTGAGCGACAGGCCCGAGGCCACCTTCCGGGCCAGGCTGGACCTCGGCATCCCCGGCGACGTGCCCAAGTACGACATCATCTTCGTGAACGTCAGGACCCCATACAAGTACCACCATTACCAGCAGTGCGAGGACCACGCCATCAAGCTGAGCATGCTGACCAAGAAGGCCTGCCTGCACCTGAACCCCGGAGGCACCTGCGTGAGCATCGGCTACGGCTACGCCGACAGGGCCAGCGAGAGCATCATTGGCGCCATCGCCAGGCTGTTCAAGTTCAGCAGGGTGTGCAAACCCAAGAGCAGCCTGGAGGAAACCGAGGTGCTGTTCGTGTTCATCGGCTACGACCGGAAGGCCAGGACCCACAACCCCTACAAGCTGAGCAGCACCCTGACAAACATCTACACCGGCAGCAGGCTGCACGAGGCCGGCTGCGCCCCCAGCTACCACGTGGTCAGGGGCGATATCGCCACCGCCACCGAGGGCGTGATCATCAACGCTGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGTGTGCGGCGCCCTGTACAAGAAGTTCCCCGAGAGCTTCGACCTGCAGCCCATCGAGGTGGGCAAGGCCAGGCTGGTGAAGGGCGCCGCTAAGCACATCATCCACGCCGTGGGCCCCAACTTCAACAAGGTGAGCGAGGTGGAAGGCGACAAGCAGCTGGCCGAAGCCTACGAGAGCATCGCCAAGATCGTGAACGACAATAACTACAAGAGCGTGGCCATCCCACTGCTCAGCACCGGCATCTTCAGCGGCAACAAGGACAGGCTGACCCAGAGCCTGAACCACCTGCTCACCGCCCTGGACACCACCGATGCCGACGTGGCCATCTACTGCAGGGACAAGAAGTGGGAGATGACCCTGAAGGAGGCCGTGGCCAGGCGGGAGGCCGTGGAAGAGATCTGCATCAGCGACGACTCCAGCGTGACCGAGCCCGACGCCGAGCTGGTGAGGGTGCACCCCAAGAGCTCCCTGGCCGGCAGGAAGGGCTACAGCACCAGCGACGGCAAGACCTTCAGCTACCTGGAGGGCACCAAGTTCCACCAGGCCGCTAAGGACATCGCCGAGATCAACGCTATGTGGCCCGTGGCCACCGAGGCCAACGAGCAGGTGTGCATGTACATCCTGGGCGAGAGCATGTCCAGCATCAGGAGCAAGTGCCCCGTGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCTGCCCTGCCTGTGCATCCACGCTATGACACCCGAGAGGGTGCAGCGGCTGAAGGCCAGCAGGCCCGAGCAGATCACCGTGTGCAGCTCCTTCCCACTGCCCAAGTACAGGATCACCGGCGTGCAGAAGATCCAGTGCAGCCAGCCCATCCTGTTCAGCCCAAAGGTGCCCGCCTACATCCACCCCAGGAAGTACCTGGTGGAGACCCCACCCGTGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCTGATCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCATCATTATCGAGGAAGAGGAAGAGGACAGCATCAGCCTGCTGAGCGACGGCCCCACCCACCAGGTGCTGCAGGTGGAGGCCGACATCCACGGCCCACCCAGCGTGTCCAGCTCCAGCTGGAGCATCCCACACGCCAGCGACTTCGACGTGGACAGCCTGAGCATCCTGGACACCCTGGAGGGCGCCAGCGTGACCTCCGGCGCCACCAGCGCCGAGACCAACAGCTACTTCGCCAAGAGCATGGAGTTCCTGGCCAGGCCCGTGCCAGCTCCCAGGACCGTGTTCAGGAACCCACCCCACCCAGCTCCCAGGACCAGGACCCCAAGCCTGGCTCCCAGCAGGGCCTGCAGCAGGACCAGCCTGGTGAGCACCCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAACTGGAGGCCCTGACACCCAGCAGGACCCCCAGCAGGTCCGTGAGCAGGACTAGTCTGGTGTCCAACCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAATTCGAGGCCTTCGTGGCCCAGCAACAGAGACGGTTCGACGCCGGCGCCTACATCTTCAGCAGCGACACCGGCCAGGGACACCTGCAGCAAAAGAGCGTGAGGCAGACCGTGCTGAGCGAGGTGGTGCTGGAGAGGACCGAGCTGGAAATCAGCTACGCCCCCAGGCTGGACCAGGAGAAGGAGGAACTGCTCAGGAAGAAACTGCAGCTGAACCCCACCCCAGCCAACAGGAGCAGGTACCAGAGCAGGAAGGTGGAGAACATGAAGGCCATCACCGCCAGGCGGATCCTGCAGGGCCTGGGACACTACCTGAAGGCCGAGGGCAAGGTGGAGTGCTACAGGACCCTGCACCCCGTGCCACTGTACAGCTCCAGCGTGAACAGGGCCTTCTCCAGCCCCAAGGTGGCCGTGGAGGCCTGCAACGCTATGCTGAAGGAGAACTTCCCCACCGTGGCCAGCTACTGCATCATCCCCGAGTACGACGCCTACCTGGACATGGTGGACGGCGCCAGCTGCTGCCTGGACACCGCCAGCTTCTGCCCCGCCAAGCTGAGGAGCTTCCCCAAGAAACACAGCTACCTGGAGCCCACCATCAGGAGCGCCGTGCCCAGCGCCATCCAGAACACCCTGCAGAACGTGCTGGCCGCTGCCACCAAGAGGAACTGCAACGTGACCCAGATGAGGGAGCTGCCCGTGCTGGACAGCGCTGCCTTCAACGTGGAGTGCTTCAAGAAATACGCCTGCAACAACGAGTACTGGGAGACCTTCAAGGAGAACCCCATCAGGCTGACCGAAGAGAACGTGGTGAACTACATCACCAAGCTGAAGGGCCCCAAGGCCGCTGCCCTGTTCGCTAAGACCCACAACCTGAACATGCTGCAGGACATCCCAATGGACAGGTTCGTGATGGACCTGAAGAGGGACGTGAAGGTGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGTGCAGGTGATCCAGGCCGCTGACCCACTGGCCACCGCCTACCTGTGCGGCATCCACAGGGAGCTGGTGAGGCGGCTGAACGCCGTGCTGCTGCCCAACATCCACACCCTGTTCGACATGAGCGCCGAGGACTTCGACGCCATCATCGCCGAGCACTTCCAGCCCGGCGACTGCGTGCTGGAGACCGACATCGCCAGCTTCGACAAGAGCGAGGATGACGCTATGGCCCTGACCGCTCTGATGATCCTGGAGGACCTGGGCGTGGACGCCGAGCTGCTCACCCTGATCGAGGCTGCCTTCGGCGAGATCAGCTCCATCCACCTGCCCACCAAGACCAAGTTCAAGTTCGGCGCTATGATGAAAAGCGGAATGTTCCTGACCCTGTTCGTGAACACCGTGATCAACATTGTGATCGCCAGCAGGGTGCTGCGGGAGAGGCTGACCGGCAGCCCCTGCGCTGCCTTCATCGGCGACGACAACATCGTGAAGGGCGTGAAAAGCGACAAGCTGATGGCCGACAGGTGCGCCACCTGGCTGAACATGGAGGTGAAGATCATCGACGCCGTGGTGGGCGAGAAGGCCCCCTACTTCTGCGGCGGATTCATCCTGTGCGACAGCGTGACCGGCACCGCCTGCAGGGTGGCCGACCCCCTGAAGAGGCTGTTCAAGCTGGGCAAGCCACTGGCCGCTGACGATGAGCACGACGATGACAGGCGGAGGGCCCTGCACGAGGAAAGCACCAGGTGGAACAGGGTGGGCATCCTGAGCGAGCTGTGCAAGGCCGTGGAGAGCAGGTACGAGACCGTGGGCACCAGCATCATCGTGATGGCTATGACCACACTGGCCAGCTCCGTCAAGAGCTTCTCCTACCTGAGGGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGGCCGCCACCACTCGAGTATGTTACGTGCAAAGGTGATTGTCACCCCCCGAAAGACCATATTGTGACACACCCTCAGTATCACGCCCAAACATTTACAGCCGCGGTGTCAAAAACCGCGTGGACGTGGTTAACATCCCTGCTGGGAGGATCAGCCGTAATTATTATAATTGGCTTGGTGCTGGCTACTATTGTGGCCATGTACGTGCTGACCAACCAGAAACATAATTGAATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAATCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 124 ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCCGTCGACGGCCCCACCAGCCTGTACCACCAGGCCAACAAGGGCGTGAGGGTGGCCTACTGGATCGGCTTCGACACCACACCCTTCATGTTCAAGAACCTGGCCGGCGCCTACCCCAGCTACAGCACCAACTGGGCCGACGAGACCGTGCTGACCGCCAGGAACATCGGCCTGTGCAGCAGCGACGTGATGGAGAGGAGCCGGAGAGGCATGAGCATCCTGAGGAAGAAATACCTGAAGCCCAGCAACAACGTGCTGTTCAGCGTGGGCAGCACCATCTACCACGAGAAGAGGGACCTGCTCAGGAGCTGGCACCTGCCCAGCGTGTTCCACCTGAGGGGCAAGCAGAACTACACCTGCAGGTGCGAGACCATCGTGAGCTGCGACGGCTACGTGGTGAAGAGGATCGCCATCAGCCCCGGCCTGTACGGCAAGCCCAGCGGCTACGCCGCTACAATGCACAGGGAGGGCTTCCTGTGCTGCAAGGTGACCGACACCCTGAACGGCGAGAGGGTGAGCTTCCCCGTGTGCACCTACGTGCCCGCCACCCTGTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGAGCGCCGACGACGCCCAGAAGCTGCTCGTGGGCCTGAACCAGAGGATCGTGGTCAACGGCAGGACCCAGAGGAACACCAACACAATGAAGAACTACCTGCTGCCCGTGGTGGCCCAGGCTTTCGCCAGGTGGGCCAAGGAGTACAAGGAGGACCAGGAAGACGAGAGGCCCCTGGGCCTGAGGGACAGGCAGCTGGTGATGGGCTGCTGCTGGGCCTTCAGGCGGCACAAGATCACCAGCATCTACAAGAGGCCCGACACCCAGACCATCATCAAGGTGAACAGCGACTTCCACAGCTTCGTGCTGCCCAGGATCGGCAGCAACACCCTGGAGATCGGCCTGAGGACCCGGATCAGGAAGATGCTGGAGGAACACAAGGAGCCCAGCCCACTGATCACCGCCGAGGACGTGCAGGAGGCCAAGTGCGCTGCCGACGAGGCCAAGGAGGTGAGGGAGGCCGAGGAACTGAGGGCCGCCCTGCCACCCCTGGCTGCCGACGTGGAGGAACCCACCCTGGAAGCCGACGTGGACCTGATGCTGCAGGAGGCCGGCGCCGGAAGCGTGGAGACACCCAGGGGCCTGATCAAGGTGACCAGCTACGACGGCGAGGACAAGATCGGCAGCTACGCCGTGCTGAGCCCACAGGCCGTGCTGAAGTCCGAGAAGCTGAGCTGCATCCACCCACTGGCCGAGCAGGTGATCGTGATCACCCACAGCGGCAGGAAGGGCAGGTACGCCGTGGAGCCCTACCACGGCAAGGTGGTCGTGCCCGAGGGCCACGCCATCCCCGTGCAGGACTTCCAGGCCCTGAGCGAGAGCGCCACCATCGTGTACAACGAGAGGGAGTTCGTGAACAGGTACCTGCACCATATCGCCACCCACGGCGGAGCCCTGAACACCGACGAGGAATACTACAAGACCGTGAAGCCCAGCGAGCACGACGGCGAGTACCTGTACGACATCGACAGGAAGCAGTGCGTGAAGAAAGAGCTGGTGACCGGCCTGGGACTGACCGGCGAGCTGGTGGACCCACCCTTCCACGAGTTCGCCTACGAGAGCCTGAGGACCAGACCCGCCGCTCCCTACCAGGTGCCCACCATCGGCGTGTACGGCGTGCCCGGCAGCGGAAAGAGCGGCATCATCAAGAGCGCCGTGACCAAGAAAGACCTGGTGGTCAGCGCCAAGAAAGAGAACTGCGCCGAGATCATCAGGGACGTGAAGAAGATGAAAGGCCTGGACGTGAACGCGCGCACCGTGGACAGCGTGCTGCTGAACGGCTGCAAGCACCCCGTGGAGACCCTGTACATCGACGAGGCCTTCGCTTGCCACGCCGGCACCCTGAGGGCCCTGATCGCCATCATCAGGCCCAAGAAAGCCGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATGATGTGCCTGAAGGTGCACTTCAACCACGAGATCTGCACCCAGGTGTTCCACAAGAGCATCAGCAGGCGGTGCACCAAGAGCGTGACCAGCGTCGTGAGCACCCTGTTCTACGACAAGAAAATGAGGACCACCAACCCCAAGGAGACCAAAATCGTGATCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCTGATCCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCAGATCGACTACAAGGGCAACGAGATCATGACCGCCGCTGCCAGCCAGGGCCTGACCAGGAAGGGCGTGTACGCCGTGAGGTACAAGGTGAACGAGAACCCACTGTACGCTCCCACCAGCGAGCACGTGAACGTGCTGCTGACCAGGACCGAGGACAGGATCGTGTGGAAGACCCTGGCCGGCGACCCCTGGATCAAGACCCTGACCGCCAAGTACCCCGGCAACTTCACCGCCACCATCGAAGAGTGGCAGGCCGAGCACGACGCCATCATGAGGCACATCCTGGAGAGGCCCGACCCCACCGACGTGTTCCAGAACAAGGCCAACGTGTGCTGGGCCAAGGCCCTGGTGCCCGTGCTGAAGACCGCCGGCATCGACATGACCACAGAGCAGTGGAACACCGTGGACTACTTCGAGACCGACAAGGCCCACAGCGCCGAGATCGTGCTGAACCAGCTGTGCGTGAGGTTCTTCGGCCTGGACCTGGACAGCGGCCTGTTCAGCGCCCCCACCGTGCCACTGAGCATCAGGAACAACCACTGGGACAACAGCCCCAGCCCAAACATGTACGGCCTGAACAAGGAGGTGGTCAGGCAGCTGAGCAGGCGGTACCCACAGCTGCCCAGGGCCGTGGCCACCGGCAGGGTGTACGACATGAACACCGGCACCCTGAGGAACTACGACCCCAGGATCAACCTGGTGCCCGTGAACAGGCGGCTGCCCCACGCCCTGGTGCTGCACCACAACGAGCACCCACAGAGCGACTTCAGCTCCTTCGTGAGCAAGCTGAAAGGCAGGACCGTGCTGGTCGTGGGCGAGAAGCTGAGCGTGCCCGGCAAGATGGTGGACTGGCTGAGCGACAGGCCCGAGGCCACCTTCCGGGCCAGGCTGGACCTCGGCATCCCCGGCGACGTGCCCAAGTACGACATCATCTTCGTGAACGTCAGGACCCCATACAAGTACCACCATTACCAGCAGTGCGAGGACCACGCCATCAAGCTGAGCATGCTGACCAAGAAGGCCTGCCTGCACCTGAACCCCGGAGGCACCTGCGTGAGCATCGGCTACGGCTACGCCGACAGGGCCAGCGAGAGCATCATTGGCGCCATCGCCAGGCTGTTCAAGTTCAGCAGGGTGTGCAAACCCAAGAGCAGCCTGGAGGAAACCGAGGTGCTGTTCGTGTTCATCGGCTACGACCGGAAGGCCAGGACCCACAACCCCTACAAGCTGAGCAGCACCCTGACAAACATCTACACCGGCAGCAGGCTGCACGAGGCCGGCTGCGCCCCCAGCTACCACGTGGTCAGGGGCGATATCGCCACCGCCACCGAGGGCGTGATCATCAACGCTGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGTGTGCGGCGCCCTGTACAAGAAGTTCCCCGAGAGCTTCGACCTGCAGCCCATCGAGGTGGGCAAGGCCAGGCTGGTGAAGGGCGCCGCTAAGCACATCATCCACGCCGTGGGCCCCAACTTCAACAAGGTGAGCGAGGTGGAAGGCGACAAGCAGCTGGCCGAAGCCTACGAGAGCATCGCCAAGATCGTGAACGACAATAACTACAAGAGCGTGGCCATCCCACTGCTCAGCACCGGCATCTTCAGCGGCAACAAGGACAGGCTGACCCAGAGCCTGAACCACCTGCTCACCGCCCTGGACACCACCGATGCCGACGTGGCCATCTACTGCAGGGACAAGAAGTGGGAGATGACCCTGAAGGAGGCCGTGGCCAGGCGGGAGGCCGTGGAAGAGATCTGCATCAGCGACGACTCCAGCGTGACCGAGCCCGACGCCGAGCTGGTGAGGGTGCACCCCAAGAGCTCCCTGGCCGGCAGGAAGGGCTACAGCACCAGCGACGGCAAGACCTTCAGCTACCTGGAGGGCACCAAGTTCCACCAGGCCGCTAAGGACATCGCCGAGATCAACGCTATGTGGCCCGTGGCCACCGAGGCCAACGAGCAGGTGTGCATGTACATCCTGGGCGAGAGCATGTCCAGCATCAGGAGCAAGTGCCCCGTGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCTGCCCTGCCTGTGCATCCACGCTATGACACCCGAGAGGGTGCAGCGGCTGAAGGCCAGCAGGCCCGAGCAGATCACCGTGTGCAGCTCCTTCCCACTGCCCAAGTACAGGATCACCGGCGTGCAGAAGATCCAGTGCAGCCAGCCCATCCTGTTCAGCCCAAAGGTGCCCGCCTACATCCACCCCAGGAAGTACCTGGTGGAGACCCCACCCGTGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCTGATCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCATCATTATCGAGGAAGAGGAAGAGGACAGCATCAGCCTGCTGAGCGACGGCCCCACCCACCAGGTGCTGCAGGTGGAGGCCGACATCCACGGCCCACCCAGCGTGTCCAGCTCCAGCTGGAGCATCCCACACGCCAGCGACTTCGACGTGGACAGCCTGAGCATCCTGGACACCCTGGAGGGCGCCAGCGTGACCTCCGGCGCCACCAGCGCCGAGACCAACAGCTACTTCGCCAAGAGCATGGAGTTCCTGGCCAGGCCCGTGCCAGCTCCCAGGACCGTGTTCAGGAACCCACCCCACCCAGCTCCCAGGACCAGGACCCCAAGCCTGGCTCCCAGCAGGGCCTGCAGCAGGACCAGCCTGGTGAGCACCCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAACTGGAGGCCCTGACACCCAGCAGGACCCCCAGCAGGTCCGTGAGCAGGACTAGTCTGGTGTCCAACCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAATTCGAGGCCTTCGTGGCCCAGCAACAGAGACGGTTCGACGCCGGCGCCTACATCTTCAGCAGCGACACCGGCCAGGGACACCTGCAGCAAAAGAGCGTGAGGCAGACCGTGCTGAGCGAGGTGGTGCTGGAGAGGACCGAGCTGGAAATCAGCTACGCCCCCAGGCTGGACCAGGAGAAGGAGGAACTGCTCAGGAAGAAACTGCAGCTGAACCCCACCCCAGCCAACAGGAGCAGGTACCAGAGCAGGAAGGTGGAGAACATGAAGGCCATCACCGCCAGGCGGATCCTGCAGGGCCTGGGACACTACCTGAAGGCCGAGGGCAAGGTGGAGTGCTACAGGACCCTGCACCCCGTGCCACTGTACAGCTCCAGCGTGAACAGGGCCTTCTCCAGCCCCAAGGTGGCCGTGGAGGCCTGCAACGCTATGCTGAAGGAGAACTTCCCCACCGTGGCCAGCTACTGCATCATCCCCGAGTACGACGCCTACCTGGACATGGTGGACGGCGCCAGCTGCTGCCTGGACACCGCCAGCTTCTGCCCCGCCAAGCTGAGGAGCTTCCCCAAGAAACACAGCTACCTGGAGCCCACCATCAGGAGCGCCGTGCCCAGCGCCATCCAGAACACCCTGCAGAACGTGCTGGCCGCTGCCACCAAGAGGAACTGCAACGTGACCCAGATGAGGGAGCTGCCCGTGCTGGACAGCGCTGCCTTCAACGTGGAGTGCTTCAAGAAATACGCCTGCAACAACGAGTACTGGGAGACCTTCAAGGAGAACCCCATCAGGCTGACCGAAGAGAACGTGGTGAACTACATCACCAAGCTGAAGGGCCCCAAGGCCGCTGCCCTGTTCGCTAAGACCCACAACCTGAACATGCTGCAGGACATCCCAATGGACAGGTTCGTGATGGACCTGAAGAGGGACGTGAAGGTGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGTGCAGGTGATCCAGGCCGCTGACCCACTGGCCACCGCCTACCTGTGCGGCATCCACAGGGAGCTGGTGAGGCGGCTGAACGCCGTGCTGCTGCCCAACATCCACACCCTGTTCGACATGAGCGCCGAGGACTTCGACGCCATCATCGCCGAGCACTTCCAGCCCGGCGACTGCGTGCTGGAGACCGACATCGCCAGCTTCGACAAGAGCGAGGATGACGCTATGGCCCTGACCGCTCTGATGATCCTGGAGGACCTGGGCGTGGACGCCGAGCTGCTCACCCTGATCGAGGCTGCCTTCGGCGAGATCAGCTCCATCCACCTGCCCACCAAGACCAAGTTCAAGTTCGGCGCTATGATGAAAAGCGGAATGTTCCTGACCCTGTTCGTGAACACCGTGATCAACATTGTGATCGCCAGCAGGGTGCTGCGGGAGAGGCTGACCGGCAGCCCCTGCGCTGCCTTCATCGGCGACGACAACATCGTGAAGGGCGTGAAAAGCGACAAGCTGATGGCCGACAGGTGCGCCACCTGGCTGAACATGGAGGTGAAGATCATCGACGCCGTGGTGGGCGAGAAGGCCCCCTACTTCTGCGGCGGATTCATCCTGTGCGACAGCGTGACCGGCACCGCCTGCAGGGTGGCCGACCCCCTGAAGAGGCTGTTCAAGCTGGGCAAGCCACTGGCCGCTGACGATGAGCACGACGATGACAGGCGGAGGGCCCTGCACGAGGAAAGCACCAGGTGGAACAGGGTGGGCATCCTGAGCGAGCTGTGCAAGGCCGTGGAGAGCAGGTACGAGACCGTGGGCACCAGCATCATCGTGATGGCTATGACCACACTGGCCAGCTCCGTCAAGAGCTTCTCCTACCTGAGGGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGGCCGCCACCATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTICAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTACACATAAACTCGAGTATGTTACGTGCAAAGGTGATTGTCACCCCCCGAAAGACCATATTGTGACACACCCTCAGTATCACGCCCAAACATTTACAGCCGCGGTGTCAAAAACCGCGTGGACGTGGTTAACATCCCTGCTGGGAGGATCAGCCGTAATTATTATAATTGGCTTGGTGCTGGCTACTATTGTGGCCATGTACGTGCTGACCAACCAGAAACATAATTGAATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAATCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 125 ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTGGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATACGCCGTCGACGGCCCCACCAGCCTGTACCACCAGGCCAACAAGGGCGTGAGGGTGGCCTACTGGATCGGCTTCGACACCACACCCTTCATGTTCAAGAACCTGGCCGGCGCCTACCCCAGCTACAGCACCAACTGGGCCGACGAGACCGTGCTGACCGCCAGGAACATCGGCCTGTGCAGCAGCGACGTGATGGAGAGGAGCCGGAGAGGCATGAGCATCCTGAGGAAGAAATACCTGAAGCCCAGCAACAACGTGCTGTTCAGCGTGGGCAGCACCATCTACCACGAGAAGAGGGACCTGCTCAGGAGCTGGCACCTGCCCAGCGTGTTCCACCTGAGGGGCAAGCAGAACTACACCTGCAGGTGCGAGACCATCGTGAGCTGCGACGGCTACGTGGTGAAGAGGATCGCCATCAGCCCCGGCCTGTACGGCAAGCCCAGCGGCTACGCCGCTACAATGCACAGGGAGGGCTTCCTGTGCTGCAAGGTGACCGACACCCTGAACGGCGAGAGGGTGAGCTTCCCCGTGTGCACCTACGTGCCCGCCACCCTGTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGAGCGCCGACGACGCCCAGAAGCTGCTCGTGGGCCTGAACCAGAGGATCGTGGTCAACGGCAGGACCCAGAGGAACACCAACACAATGAAGAACTACCTGCTGCCCGTGGTGGCCCAGGCTTTCGCCAGGTGGGCCAAGGAGTACAAGGAGGACCAGGAAGACGAGAGGCCCCTGGGCCTGAGGGACAGGCAGCTGGTGATGGGCTGCTGCTGGGCCTTCAGGCGGCACAAGATCACCAGCATCTACAAGAGGCCCGACACCCAGACCATCATCAAGGTGAACAGCGACTTCCACAGCTTCGTGCTGCCCAGGATCGGCAGCAACACCCTGGAGATCGGCCTGAGGACCCGGATCAGGAAGATGCTGGAGGAACACAAGGAGCCCAGCCCACTGATCACCGCCGAGGACGTGCAGGAGGCCAAGTGCGCTGCCGACGAGGCCAAGGAGGTGAGGGAGGCCGAGGAACTGAGGGCCGCCCTGCCACCCCTGGCTGCCGACGTGGAGGAACCCACCCTGGAAGCCGACGTGGACCTGATGCTGCAGGAGGCCGGCGCCGGAAGCGTGGAGACACCCAGGGGCCTGATCAAGGTGACCAGCTACGACGGCGAGGACAAGATCGGCAGCTACGCCGTGCTGAGCCCACAGGCCGTGCTGAAGTCCGAGAAGCTGAGCTGCATCCACCCACTGGCCGAGCAGGTGATCGTGATCACCCACAGCGGCAGGAAGGGCAGGTACGCCGTGGAGCCCTACCACGGCAAGGTGGTCGTGCCCGAGGGCCACGCCATCCCCGTGCAGGACTTCCAGGCCCTGAGCGAGAGCGCCACCATCGTGTACAACGAGAGGGAGTTCGTGAACAGGTACCTGCACCATATCGCCACCCACGGCGGAGCCCTGAACACCGACGAGGAATACTACAAGACCGTGAAGCCCAGCGAGCACGACGGCGAGTACCTGTACGACATCGACAGGAAGCAGTGCGTGAAGAAAGAGCTGGTGACCGGCCTGGGACTGACCGGCGAGCTGGTGGACCCACCCTTCCACGAGTTCGCCTACGAGAGCCTGAGGACCAGACCCGCCGCTCCCTACCAGGTGCCCACCATCGGCGTGTACGGCGTGCCCGGCAGCGGAAAGAGCGGCATCATCAAGAGCGCCGTGACCAAGAAAGACCTGGTGGTCAGCGCCAAGAAAGAGAACTGCGCCGAGATCATCAGGGACGTGAAGAAGATGAAAGGCCTGGACGTGAACGCGCGCACCGTGGACAGCGTGCTGCTGAACGGCTGCAAGCACCCCGTGGAGACCCTGTACATCGACGAGGCCTTCGCTTGCCACGCCGGCACCCTGAGGGCCCTGATCGCCATCATCAGGCCCAAGAAAGCCGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATGATGTGCCTGAAGGTGCACTTCAACCACGAGATCTGCACCCAGGTGTTCCACAAGAGCATCAGCAGGCGGTGCACCAAGAGCGTGACCAGCGTCGTGAGCACCCTGTTCTACGACAAGAAAATGAGGACCACCAACCCCAAGGAGACCAAAATCGTGATCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCTGATCCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCAGATCGACTACAAGGGCAACGAGATCATGACCGCCGCTGCCAGCCAGGGCCTGACCAGGAAGGGCGTGTACGCCGTGAGGTACAAGGTGAACGAGAACCCACTGTACGCTCCCACCAGCGAGCACGTGAACGTGCTGCTGACCAGGACCGAGGACAGGATCGTGTGGAAGACCCTGGCCGGCGACCCCTGGATCAAGACCCTGACCGCCAAGTACCCCGGCAACTTCACCGCCACCATCGAAGAGTGGCAGGCCGAGCACGACGCCATCATGAGGCACATCCTGGAGAGGCCCGACCCCACCGACGTGTTCCAGAACAAGGCCAACGTGTGCTGGGCCAAGGCCCTGGTGCCCGTGCTGAAGACCGCCGGCATCGACATGACCACAGAGCAGTGGAACACCGTGGACTACTTCGAGACCGACAAGGCCCACAGCGCCGAGATCGTGCTGAACCAGCTGTGCGTGAGGTTCTTCGGCCTGGACCTGGACAGCGGCCTGTTCAGCGCCCCCACCGTGCCACTGAGCATCAGGAACAACCACTGGGACAACAGCCCCAGCCCAAACATGTACGGCCTGAACAAGGAGGTGGTCAGGCAGCTGAGCAGGCGGTACCCACAGCTGCCCAGGGCCGTGGCCACCGGCAGGGTGTACGACATGAACACCGGCACCCTGAGGAACTACGACCCCAGGATCAACCTGGTGCCCGTGAACAGGCGGCTGCCCCACGCCCTGGTGCTGCACCACAACGAGCACCCACAGAGCGACTTCAGCTCCTTCGTGAGCAAGCTGAAAGGCAGGACCGTGCTGGTCGTGGGCGAGAAGCTGAGCGTGCCCGGCAAGATGGTGGACTGGCTGAGCGACAGGCCCGAGGCCACCTTCCGGGCCAGGCTGGACCTCGGCATCCCCGGCGACGTGCCCAAGTACGACATCATCTTCGTGAACGTCAGGACCCCATACAAGTACCACCATTACCAGCAGTGCGAGGACCACGCCATCAAGCTGAGCATGCTGACCAAGAAGGCCTGCCTGCACCTGAACCCCGGAGGCACCTGCGTGAGCATCGGCTACGGCTACGCCGACAGGGCCAGCGAGAGCATCATTGGCGCCATCGCCAGGCTGTTCAAGTTCAGCAGGGTGTGCAAACCCAAGAGCAGCCTGGAGGAAACCGAGGTGCTGTTCGTGTTCATCGGCTACGACCGGAAGGCCAGGACCCACAACCCCTACAAGCTGAGCAGCACCCTGACAAACATCTACACCGGCAGCAGGCTGCACGAGGCCGGCTGCGCCCCCAGCTACCACGTGGTCAGGGGCGATATCGCCACCGCCACCGAGGGCGTGATCATCAACGCTGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGTGTGCGGCGCCCTGTACAAGAAGTTCCCCGAGAGCTTCGACCTGCAGCCCATCGAGGTGGGCAAGGCCAGGCTGGTGAAGGGCGCCGCTAAGCACATCATCCACGCCGTGGGCCCCAACTTCAACAAGGTGAGCGAGGTGGAAGGCGACAAGCAGCTGGCCGAAGCCTACGAGAGCATCGCCAAGATCGTGAACGACAATAACTACAAGAGCGTGGCCATCCCACTGCTCAGCACCGGCATCTTCAGCGGCAACAAGGACAGGCTGACCCAGAGCCTGAACCACCTGCTCACCGCCCTGGACACCACCGATGCCGACGTGGCCATCTACTGCAGGGACAAGAAGTGGGAGATGACCCTGAAGGAGGCCGTGGCCAGGCGGGAGGCCGTGGAAGAGATCTGCATCAGCGACGACTCCAGCGTGACCGAGCCCGACGCCGAGCTGGTGAGGGTGCACCCCAAGAGCTCCCTGGCCGGCAGGAAGGGCTACAGCACCAGCGACGGCAAGACCTTCAGCTACCTGGAGGGCACCAAGTTCCACCAGGCCGCTAAGGACATCGCCGAGATCAACGCTATGTGGCCCGTGGCCACCGAGGCCAACGAGCAGGTGTGCATGTACATCCTGGGCGAGAGCATGTCCAGCATCAGGAGCAAGTGCCCCGTGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCTGCCCTGCCTGTGCATCCACGCTATGACACCCGAGAGGGTGCAGCGGCTGAAGGCCAGCAGGCCCGAGCAGATCACCGTGTGCAGCTCCTTCCCACTGCCCAAGTACAGGATCACCGGCGTGCAGAAGATCCAGTGCAGCCAGCCCATCCTGTTCAGCCCAAAGGTGCCCGCCTACATCCACCCCAGGAAGTACCTGGTGGAGACCCCACCCGTGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCTGATCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCATCATTATCGAGGAAGAGGAAGAGGACAGCATCAGCCTGCTGAGCGACGGCCCCACCCACCAGGTGCTGCAGGTGGAGGCCGACATCCACGGCCCACCCAGCGTGTCCAGCTCCAGCTGGAGCATCCCACACGCCAGCGACTTCGACGTGGACAGCCTGAGCATCCTGGACACCCTGGAGGGCGCCAGCGTGACCTCCGGCGCCACCAGCGCCGAGACCAACAGCTACTTCGCCAAGAGCATGGAGTTCCTGGCCAGGCCCGTGCCAGCTCCCAGGACCGTGTTCAGGAACCCACCCCACCCAGCTCCCAGGACCAGGACCCCAAGCCTGGCTCCCAGCAGGGCCTGCAGCAGGACCAGCCTGGTGAGCACCCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAACTGGAGGCCCTGACACCCAGCAGGACCCCCAGCAGGTCCGTGAGCAGGACTAGTCTGGTGTCCAACCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAATTCGAGGCCTTCGTGGCCCAGCAACAGAGACGGTTCGACGCCGGCGCCTACATCTTCAGCAGCGACACCGGCCAGGGACACCTGCAGCAAAAGAGCGTGAGGCAGACCGTGCTGAGCGAGGTGGTGCTGGAGAGGACCGAGCTGGAAATCAGCTACGCCCCCAGGCTGGACCAGGAGAAGGAGGAACTGCTCAGGAAGAAACTGCAGCTGAACCCCACCCCAGCCAACAGGAGCAGGTACCAGAGCAGGAAGGTGGAGAACATGAAGGCCATCACCGCCAGGCGGATCCTGCAGGGCCTGGGACACTACCTGAAGGCCGAGGGCAAGGTGGAGTGCTACAGGACCCTGCACCCCGTGCCACTGTACAGCTCCAGCGTGAACAGGGCCTTCTCCAGCCCCAAGGTGGCCGTGGAGGCCTGCAACGCTATGCTGAAGGAGAACTTCCCCACCGTGGCCAGCTACTGCATCATCCCCGAGTACGACGCCTACCTGGACATGGTGGACGGCGCCAGCTGCTGCCTGGACACCGCCAGCTTCTGCCCCGCCAAGCTGAGGAGCTTCCCCAAGAAACACAGCTACCTGGAGCCCACCATCAGGAGCGCCGTGCCCAGCGCCATCCAGAACACCCTGCAGAACGTGCTGGCCGCTGCCACCAAGAGGAACTGCAACGTGACCCAGATGAGGGAGCTGCCCGTGCTGGACAGCGCTGCCTTCAACGTGGAGTGCTTCAAGAAATACGCCTGCAACAACGAGTACTGGGAGACCTTCAAGGAGAACCCCATCAGGCTGACCGAAGAGAACGTGGTGAACTACATCACCAAGCTGAAGGGCCCCAAGGCCGCTGCCCTGTTCGCTAAGACCCACAACCTGAACATGCTGCAGGACATCCCAATGGACAGGTTCGTGATGGACCTGAAGAGGGACGTGAAGGTGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGTGCAGGTGATCCAGGCCGCTGACCCACTGGCCACCGCCTACCTGTGCGGCATCCACAGGGAGCTGGTGAGGCGGCTGAACGCCGTGCTGCTGCCCAACATCCACACCCTGTTCGACATGAGCGCCGAGGACTTCGACGCCATCATCGCCGAGCACTTCCAGCCCGGCGACTGCGTGCTGGAGACCGACATCGCCAGCTTCGACAAGAGCGAGGATGACGCTATGGCCCTGACCGCTCTGATGATCCTGGAGGACCTGGGCGTGGACGCCGAGCTGCTCACCCTGATCGAGGCTGCCTTCGGCGAGATCAGCTCCATCCACCTGCCCACCAAGACCAAGTTCAAGTTCGGCGCTATGATGAAAAGCGGAATGTTCCTGACCCTGTTCGTGAACACCGTGATCAACATTGTGATCGCCAGCAGGGTGCTGCGGGAGAGGCTGACCGGCAGCCCCTGCGCTGCCTTCATCGGCGACGACAACATCGTGAAGGGCGTGAAAAGCGACAAGCTGATGGCCGACAGGTGCGCCACCTGGCTGAACATGGAGGTGAAGATCATCGACGCCGTGGTGGGCGAGAAGGCCCCCTACTTCTGCGGCGGATTCATCCTGTGCGACAGCGTGACCGGCACCGCCTGCAGGGTGGCCGACCCCCTGAAGAGGCTGTTCAAGCTGGGCAAGCCACTGGCCGCTGACGATGAGCACGACGATGACAGGCGGAGGGCCCTGCACGAGGAAAGCACCAGGTGGAACAGGGTGGGCATCCTGAGCGAGCTGTGCAAGGCCGTGGAGAGCAGGTACGAGACCGTGGGCACCAGCATCATCGTGATGGCTATGACCACACTGGCCAGCTCCGTCAAGAGCTTCTCCTACCTGAGGGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGGCCGCCACCATGTTCGTCTTCCTGGTCCTGCTGCCTCTGGTCTCCTCACAGTGCGTCAATCTGACAACTCGGACTCAGCTGCCACCTGCTTATACTAATAGCTTCACCAGAGGCGTGTACTATCCTGACAAGGTGTTTAGAAGCTCCGTGCTGCACTCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAATGGCACAAAGCGGTTCGACAATCCCGTGCTGCCTTTTAACGATGGCGTGTACTTCGCCTCTACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTTGGCACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGTCATCAAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAATAAGAGCTGGATGGAGTCCGAGTTTAGAGTGTATTCTAGCGCCAACAACTGCACATTTGAGTACGTGAGCCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGACGGCTACTTCAAAATCTACTCTAAGCACACCCCCATCAACCTGGTGCGCGACCTGCCTCAGGGCTTCAGCGCCCTGGAGCCCCTGGTGGATCTGCCTATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAGCGGATGGACCGCCGGCGCTGCCGCCTACTATGTGGGCTACCTCCAGCCCCGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATCACAGACGCAGTGGATTGCGCCCTGGACCCCCTGAGCGAGACAAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATCAGACATCCAATTTCAGGGTGCAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGTTCAACGCAACCCGCTTCGCCAGCGTGTACGCCTGGAATAGGAAGCGGATCAGCAACTGCGTGGCCGACTATAGCGTGCTGTACAACTCCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACCTGTGCTTTACCAACGTCTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGCCCCCGGCCAGACAGGCAAGATCGCAGACTACAATTATAAGCTGCCAGACGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGGCAACTACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCTACAGAAATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCCACTCCAGTCCTACGGCTTCCAGCCCACAAACGGCGTGGGCTATCAGCCTTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCCCCAGCAACAGTGTGCGGCCCCAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGCACAGGCGTGCTGACCGAGTCCAACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGACCCACAGACCCTGGAGATCCTGGACATCACACCCTGCTCTTTCGGCGGCGTGAGCGTGATCACACCCGGCACCAATACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGTACCGAGGTGCCCGTGGCTATCCACGCCGATCAGCTGACCCCAACATGGCGGGTGTACAGCACCGGCTCCAACGTCTTCCAGACAAGAGCCGGATGCCTGATCGGAGCAGAGCACGTGAACAATTCCTATGAGTGCGACATCCCAATCGGCGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCCAGACGGGCCCGGAGCGTGGCCTCCCAGTCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAACAGCGTGGCCTACTCTAACAATAGCATCGCCATCCCAACCAACTTCACAATCTCTGTGACCACAGAGATCCTGCCCGTGTCCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTACCGAGTGCAGCAACCTGCTGCTCCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCCCTGACAGGCATCGCCGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAAATCTACAAGACCCCCCCTATCAAGGACTTTGGCGGCTTCAATTTTTCCCAGATCCTGCCTGATCCATCCAAGCCTTCTAAGCGGAGCTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCAGCCAGGGACCTGATCTGCGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCACCCCTGCTGACAGATGAGATGATCGCACAGTACACAAGCGCCCTGCTGGCCGGCACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTCCAGATCCCCTTTGCCATGCAGATGGCCTATAGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCTGTCCTCTACAGCCAGCGCCCTGGGCAAGCTCCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCATCTCTAGCGTGCTGAATGACATCCTGAGCCGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACCGGCCGGCTCCAGAGCCTCCAGACCTATGTGACACAGCAGCTGATCAGGGCCGCCGAGATCAGGGCCAGCGCCAATCTGGCAGCAACCAAGATGTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGGGCTATCACCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTGGTGTTTCTGCACGTGACCTACGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAAGGCCCACTTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCTACGAGCCCCAGATCATCACCACAGACAACACCTTCGTGAGCGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCACTCCAGCCCGAGCTGGACAGCTTTAAGGAGGAGCTGGATAAGTATTTCAAGAATCACACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCTAAGAATCTGAACGAGAGCCTGATCGACCTCCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGACATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGACGAGGATGACTCTGAACCTGTGCTGAAGGGCGTGAAGCTGCATTACACCTAAACTCGAGTATGTTACGTGCAAAGGTGATTGTCACCCCCCGAAAGACCATATTGTGACACACCCTCAGTATCACGCCCAAACATTTACAGCCGCGGTGTCAAAAACCGCGTGGACGTGGTTAACATCCCTGCTGGGAGGATCAGCCGTAATTATTATAATTGGCTTGGTGCTGGCTACTATTGTGGCCATGTACGTGCTGACCAACCAGAAACATAATTGAATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAATCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 79 MEKVHVDIEEDSPFLRALQRSFPQFEVEAKQVTDNDHANARAFSHLASKLIETEVDPSDTILDIGSAPARRMYSKHKYHCICPMRCAEDPDRLYKYATKLKKNCKEITDKELDKKMKELAAVMSDPDLETETMCLHDDESCRYEGQVAVYQDVYAVDGPTSLYHQANKGVRVAYWIGFDTTPFMFKNLAGAYPSYSTNWADETVLTARNIGLCSSDVMERSRRGMSILRKKYLKPSNNVLFSVGSTIYHEKRDLLRSWHLPSVFHLRGKQNYTCRCETIVSCDGYVVKRIAISPGLYGKPSGYAATMHREGFLCCKVTDTLNGERVSFPVCTYVPATLCDQMTGILATDVSADDAQKLLVGLNQRIVVNGRTQRNTNTMKNYLLPVVAQAFARWAKEYKEDQEDERPLGLRDRQLVMGCCWAFRRHKITSIYKRPDTQTIIKVNSDFHSFVLPRIGSNTLEIGLRTRIRKMLEEHKEPSPLITAEDVQEAKCAADEAKEVREAEELRAALPPLAADVEEPTLEADVDLMLQEAGAGSVETPRGLIKVTSYDGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARLFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGCAPSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPINIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVTSGATSAETNSYFAKSMEFLARPVPAPRTVFRNPPHPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITREELEALTPSRTPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQRRFDAGAYIFSSDTGQGHLQQKSVRQTVLSEVVLERTELEISYAPRLDQEKEELLRKKLQLNPTPANRSRYQSRKVENMKAITARRILQGLGHYLKAEGKVECYRTLHPVPLYSSSVNRAFSSPKVAVEACNAMLKENFPTVASYCIIPEYDAYLDMVDGASCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGPKAAALFAKTHNLNMLQDIPMDRFVMDLKRDVKVTPGTKHTEERPKVQVIQAADPLATAYLCGIHRELVRRLNAVLLPNIHTLFDMSAEDFDAIIAEHFQPGDCVLETDIASFDKSEDDAMALTALMILEDLGVDAELLTLIEAAFGEISSIHLPTKTKFKFGAMMKSGMFLTLFVNTVINIVIASRVLRERLTGSPCAAFIGDDNIVKGVKSDKLMADRCATWLNMEVKIIDAVVGEKAPYFCGGFILCDSVTGTACRVADPLKRLFKLGKPLAADDEHDDDRRRALHEESTRWNRVGILSELCKAVESRYETVGTSIIVMAMTTLASSVKSFSYLRGAPITLYG

SEQ ID NO: 80 MPEKVHVDIEEDSPFLRALQRSFPQFEVEAKQVTDNDHANARAFSHLASKLIETEVDPSDTILDIGSAPARRMYSKHKYHCICPMRCAEDPDRLYKYATKLKKNCKEITDKELDKKMKELAAVMSDPDLETETMCLHDDESCRYEGQVAVYQDVYAVDGPTSLYHQANKGVRVAYWIGFDTTPFMFKNLAGAYPSYSTNWADETVLTARNIGLCSSDVMERSRRGMSILRKKYLKPSNNVLFSVGSTIYHEKRDLLRSWHLPSVFHLRGKQNYTCRCETIVSCDGYVVKRIAISPGLYGKPSGYAATMHREGFLCCKVTDTLNGERVSFPVCTYVPATLCDQMTGILATDVSADDAQKLLVGLNQRIVVNGRTQRNTNTMKNYLLPVVAQAFARWAKEYKEDQEDERPLGLRDRQLVMGCCWAFRRHKITSIYKRPDTQTIIKVNSDFHSFVLPRIGSNTLEIGLRTRIRKMLEEHKEPSPLITAEDVQEAKCAADEAKEVREAEELRAALPPLAADVEEPTLEADVDLMLQEAGAGSVETPRGLIKVTSYDGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKMVDWLSDRPEATFRARLDLGIPGDVPKYDIIFVNVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARLFKFSRVCKPKSSLEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGCAPSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVTSGATSAETNSYFAKSMEFLARPVPAPRTVFRNPPHPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITTGQGHLQQKSVRQTVLSEVVLERTELEISYAPRLDQEKEELLRKKLQLNPTPANRSRYQSRKVENMKAITARRILQGLGHYLKAEGKVECYRTLHPVPLYSSSVNRAFSSPKVAVEACNAMLKENFPTVASYCHIPEYDAYLDMVDGASCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGPKAAALFAKTHNLNMLQDIPMDRFVMDLKRDVKVTPGTKHTEERPKVQVIQAADPLATAYLCGIHRELVRRLNAVLLPNIHTLFDMAFGEISSIHLPTKTKFKFGAMMKSGMFLTLFVNTVINIVIASRVLRERLTGSPCAAFIGDDNIVKGVKSDKLMADRCATWLNMEVKIIDAVVGEKAPYFCGGFILCDSVTGTACRVADPLKRLFKLGKPLAADDEHDDDRRRALHEESTRWNRVGILSELCKAVESRYETVGTSIIVMAMTTLASSVKSFSYLRGAPITLYG

SEQ ID NO: 81 MEKVHVDIEEDSPFLRALQRSFPQFEVEAKQVTDNDHANARAFSHLASKLIETEVDPSDTILDIGSAPARRMYSKHKYHCICPMRCAEDPDRLYKYATKLKKNCKEITDKELDKKMKELAAVMSDPDLETETMCLHDDESCRYEGQVAVYQDVYAVDGPTSLYHQANKGVRVAYWIGFDTTPFMFKNLAGAYPSYSTNWADETVLTARNIGLCSSDVMERSRRGMSILRKKYLKPSNNVLFSVGSTIYHEKRDLLRSWHLPSVFHLRGKQNYTCRCETIVSCDGYVVKRIAISPGLYGKPSGYAATMHREGFLCCKVTDTLNGERVSFPVCTYVPATLCDQMTGILATDVSADDAQKLLVGLNQRIVVNGRTQRNTNTMKNYLLPVVAQAFARWAKEYKEDQEDERPLGLRDRQLVMGCCWAFRRHKITSIYKRPDTQTIIKVNSDFHSFVLPRIGSNTLEIGLRTRIRKMLEEHKEPSPLITAEDIQEAKCAADEAKEVREAEELRAALPPLAADFEEPTLEADVDLMLQEAGAGSVETPRGLIKVTSYAGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKRMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKKVDWLSDQPEATFRARLDLGIPGDVPKYDIVFINVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSHEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGCAPSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVEETPESPAENQSTEGTPEQPALVNVDATRTRMPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGSPSVSSSSWSIPHASDFDVDSLSILDTLDGASVTSGAVSAETNSYFARSMEFRARPVPAPRTVFRNPPHPAPRTRTPPLAHSRASSRTSLVSTPPGVNRVITREELEALTPSRAPSRSASRTSLVSNPPGVNRVITREEFEAFVAQQQ*RFDAGAYIFSSDTGQGHLQQKSVRQTVLSEVVLERTELEISYAPRLDQEKEELLRKKLQLNPTPANRSRYQSRRVENMKAITARRILQGLGHYLKAEGKVECYRTLHPVPLYSSSVNRAFSSPKVAVEACNAMLKENFPTVASYCIIPEYDAYLDMVDGASCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGPKAAALFAKTHNLNMLQDIPMDRFVMDLKRDVKVTPGTKHTEERPKVQVIQAADPLATADLCGIHRELVRRLNAVLLPNIHTLFDMSAEDFDAIIAEHFQPGDCVLETDIASFDKSEDDAMALTALMILEDLGVDAELLTLIEAAFGEISSIHLPTKTKFKFGAMMKSGMFLTLFVNTVINIVIASRVLRERLTGSPCAAFIGDDNIVKGVKSDKLMADRCATWLNMEVKIIDAVVGEKAPYFCGGFILCDSVTGTACRVADPLKRLFKLGKPLAVDDEHDDDRRRALHEESTRWNRVGILPELCKAVESRYETVGTSIIVMAMTTLASSVKSFSYLRGAPITLYG*

SEQ ID NO: 126 AGGAAACTTAAGTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCCACCATGTTCGTCTTCCTGGTCCTGCTGCCTCTGGTCTCCTCACAGTGCGTCAATCTGACAACTCGGACTCAGCTGCCACCTGCTTATACTAATAGCTTCACCAGAGGCGTGTACTATCCTGACAAGGTGTTTAGAAGCTCCGTGCTGCACTCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAATGGCACAAAGCGGTTCGACAATCCCGTGCTGCCTTTTAACGATGGCGTGTACTTCGCCTCTACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTTGGCACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGTCATCAAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAATAAGAGCTGGATGGAGTCCGAGTTTAGAGTGTATTCTAGCGCCAACAACTGCACATTTGAGTACGTGAGCCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGACGGCTACTTCAAAATCTACTCTAAGCACACCCCCATCAACCTGGTGCGCGACCTGCCTCAGGGCTTCAGCGCCCTGGAGCCCCTGGTGGATCTGCCTATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAGCGGATGGACCGCCGGCGCTGCCGCCTACTATGTGGGCTACCTCCAGCCCCGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATCACAGACGCAGTGGATTGCGCCCTGGACCCCCTGAGCGAGACAAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATCAGACATCCAATTTCAGGGTGCAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGTTCAACGCAACCCGCTTCGCCAGCGTGTACGCCTGGAATAGGAAGCGGATCAGCAACTGCGTGGCCGACTATAGCGTGCTGTACAACTCCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACCTGTGCTTTACCAACGTCTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGCCCCCGGCCAGACAGGCAAGATCGCAGACTACAATTATAAGCTGCCAGACGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGGCAACTACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCTACAGAAATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCCACTCCAGTCCTACGGCTTCCAGCCCACAAACGGCGTGGGCTATCAGCCTTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCCCCAGCAACAGTGTGCGGCCCCAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGCACAGGCGTGCTGACCGAGTCCAACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGACCCACAGACCCTGGAGATCCTGGACATCACACCCTGCTCTTTCGGCGGCGTGAGCGTGATCACACCCGGCACCAATACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGTACCGAGGTGCCCGTGGCTATCCACGCCGATCAGCTGACCCCAACATGGCGGGTGTACAGCACCGGCTCCAACGTCTTCCAGACAAGAGCCGGATGCCTGATCGGAGCAGAGCACGTGAACAATTCCTATGAGTGCGACATCCCAATCGGCGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCCAGACGGGCCCGGAGCGTGGCCTCCCAGTCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAACAGCGTGGCCTACTCTAACAATAGCATCGCCATCCCAACCAACTTCACAATCTCTGTGACCACAGAGATCCTGCCCGTGTCCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTACCGAGTGCAGCAACCTGCTGCTCCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCCCTGACAGGCATCGCCGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAAATCTACAAGACCCCCCCTATCAAGGACTTTGGCGGCTTCAATTTTTCCCAGATCCTGCCTGATCCATCCAAGCCTTCTAAGCGGAGCTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCAGCCAGGGACCTGATCTGCGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCACCCCTGCTGACAGATGAGATGATCGCACAGTACACAAGCGCCCTGCTGGCCGGCACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTCCAGATCCCCTTTGCCATGCAGATGGCCTATAGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCTGTCCTCTACAGCCAGCGCCCTGGGCAAGCTCCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCATCTCTAGCGTGCTGAATGACATCCTGAGCCGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACCGGCCGGCTCCAGAGCCTCCAGACCTATGTGACACAGCAGCTGATCAGGGCCGCCGAGATCAGGGCCAGCGCCAATCTGGCAGCAACCAAGATGTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGGGCTATCACCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTGGTGTTTCTGCACGTGACCTACGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAAGGCCCACTTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCTACGAGCCCCAGATCATCACCACAGACAACACCTTCGTGAGCGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCACTCCAGCCCGAGCTGGACAGCTTTAAGGAGGAGCTGGATAAGTATTTCAAGAATCACACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCTAAGAATCTGAACGAGAGCCTGATCGACCTCCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGACATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGACGAGGATGACTCTGAACCTGTGCTGAAGGGCGTGAAGCTGCATTACACCTAAACTCGAGCTAGTGACTGACTAGGATCTGGTTACCACTAAACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 82 AGGAAACTTAAGTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAG CCACC

SEQ ID NO: 83 ACTCGAGCTAGTGACTGACTAGGATCTGGTTACCACTAAACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 1 GAGGAAACTT AAGAUGGG

SEQ ID NO:2 GGAUGGG

SEQ ID NO:3 GGAUAGG

SEQ ID NO:4 GGAGAGG

SEQ ID NO:58 GGGAUGGG

SEQ ID NO:59 GAGAGG

SEQ ID NO:60 GAGGG

SEQ ID NO:61 GAGAUGGG

SEQ ID NO:62 GAGUGG

SEQ ID NO:63 GAGGGG

SEQ ID NO:64 GAGUAGG

SEQ ID NO:65 GAGUGGG

(RNA sequence for a construct with two subgenomic promoters, Luc, and E3L)SEQ ID NO:127 atgggoggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttgacatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaactgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcagaatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctggccgccgtcatgagcgaccctgacctggaaactgagactatgtgcctccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcCGTCGACGGCCCCACCAGCCTGTACCACCAGGCCAACAAGGGCGTGAGGGTGGCCTACTGGATCGGCTTCGACACCACACCCTTCATGTTCAAGAACCTGGCCGGCGCCTACCCCAGCTACAGCACCAACTGGGCCGACGAGACCGTGCTGACCGCCAGGAACATCGGCCTGTGCAGCAGCGACGTGATGGAGAGGAGCCGGAGAGGCATGAGCATCCTGAGGAAGAAATACCTGAAGCCCAGCAACAACGTGCTGTTCAGCGTGGGCAGCACCATCTACCACGAGAAGAGGGACCTGCTCAGGAGCTGGCACCTGCCCAGCGTGTTCCACCTGAGGGGCAAGCAGAACTACACCTGCAGGTGCGAGACCATCGTGAGCTGCGACGGCTACGTGGTGAAGAGGATCGCCATCAGCCCCGGCCTGTACGGCAAGCCCAGCGGCTACGCCGCTACAATGCACAGGGAGGGCTTCCTGTGCTGCAAGGTGACCGACACCCTGAACGGCGAGAGGGTGAGCTTCCCCGTGTGCACCTACGTGCCCGCCACCCTGTGCGACCAGATGACCGGCATCCTGGCCACCGACGTGAGCGCCGACGACGCCCAGAAGCTGCTCGTGGGCCTGAACCAGAGGATCGTGGTCAACGGCAGGACCCAGAGGAACACCAACACAATGAAGAACTACCTGCTGCCCGTGGTGGCCCAGGCTTTCGCCAGGTGGGCCAAGGAGTACAAGGAGGACCAGGAAGACGAGAGGCCCCTGGGCCTGAGGGACAGGCAGCTGGTGATGGGCTGCTGCTGGGCCTTCAGGCGGCACAAGATCACCAGCATCTACAAGAGGCCCGACACCCAGACCATCATCAAGGTGAACAGCGACTTCCACAGCTTCGTGCTGCCCAGGATCGGCAGCAACACCCTGGAGATCGGCCTGAGGACCCGGATCAGGAAGATGCTGGAGGAACACAAGGAGCCCAGCCCACTGATCACCGCCGAGGACGTGCAGGAGGCCAAGTGCGCTGCCGACGAGGCCAAGGAGGTGAGGGAGGCCGAGGAACTGAGGGCCGCCCTGCCACCCCTGGCTGCCGACGTGGAGGAACCCACCCTGGAAGCCGACGTGGACCTGATGCTGCAGGAGGCCGGCGCCGGAAGCGTGGAGACACCCAGGGGCCTGATCAAGGTGACCAGCTACGACGGCGAGGACAAGATCGGCAGCTACGCCGTGCTGAGCCCACAGGCCGTGCTGAAGTCCGAGAAGCTGAGCTGCATCCACCCACTGGCCGAGCAGGTGATCGTGATCACCCACAGCGGCAGGAAGGGCAGGTACGCCGTGGAGCCCTACCACGGCAAGGTGGTCGTGCCCGAGGGCCACGCCATCCCCGTGCAGGACTTCCAGGCCCTGAGCGAGAGCGCCACCATCGTGTACAACGAGAGGGAGTTCGTGAACAGGTACCTGCACCATATCGCCACCCACGGCGGAGCCCTGAACACCGACGAGGAATACTACAAGACCGTGAAGCCCAGCGAGCACGACGGCGAGTACCTGTACGACATCGACAGGAAGCAGTGCGTGAAGAAAGAGCTGGTGACCGGCCTGGGACTGACCGGCGAGCTGGTGGACCCACCCTTCCACGAGTTCGCCTACGAGAGCCTGAGGACCAGACCCGCCGCTCCCTACCAGGTGCCCACCATCGGCGTGTACGGCGTGCCCGGCAGCGGAAAGAGCGGCATCATCAAGAGCGCCGTGACCAAGAAAGACCTGGTGGTCAGCGCCAAGAAAGAGAACTGCGCCGAGATCATCAGGGACGTGAAGAAGATGAAAGGCCTGGACGTGAACGCGCGCACCGTGGACAGCGTGCTGCTGAACGGCTGCAAGCACCCCGTGGAGACCCTGTACATCGACGAGGCCTTCGCTTGCCACGCCGGCACCCTGAGGGCCCTGATCGCCATCATCAGGCCCAAGAAAGCCGTGCTGTGCGGCGACCCCAAGCAGTGCGGCTTCTTCAACATGATGTGCCTGAAGGTGCACTTCAACCACGAGATCTGCACCCAGGTGTTCCACAAGAGCATCAGCAGGCGGTGCACCAAGAGCGTGACCAGCGTCGTGAGCACCCTGTTCTACGACAAGAAAATGAGGACCACCAACCCCAAGGAGACCAAAATCGTGATCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCTGATCCTGACCTGCTTCAGGGGCTGGGTGAAGCAGCTGCAGATCGACTACAAGGGCAACGAGATCATGACCGCCGCTGCCAGCCAGGGCCTGACCAGGAAGGGCGTGTACGCCGTGAGGTACAAGGTGAACGAGAACCCACTGTACGCTCCCACCAGCGAGCACGTGAACGTGCTGCTGACCAGGACCGAGGACAGGATCGTGTGGAAGACCCTGGCCGGCGACCCCTGGATCAAGACCCTGACCGCCAAGTACCCCGGCAACTTCACCGCCACCATCGAAGAGTGGCAGGCCGAGCACGACGCCATCATGAGGCACATCCTGGAGAGGCCCGACCCCACCGACGTGTTCCAGAACAAGGCCAACGTGTGCTGGGCCAAGGCCCTGGTGCCCGTGCTGAAGACCGCCGGCATCGACATGACCACAGAGCAGTGGAACACCGTGGACTACTTCGAGACCGACAAGGCCCACAGCGCCGAGATCGTGCTGAACCAGCTGTGCGTGAGGTTCTTCGGCCTGGACCTGGACAGCGGCCTGTTCAGCGCCCCCACCGTGCCACTGAGCATCAGGAACAACCACTGGGACAACAGCCCCAGCCCAAACATGTACGGCCTGAACAAGGAGGTGGTCAGGCAGCTGAGCAGGCGGTACCCACAGCTGCCCAGGGCCGTGGCCACCGGCAGGGTGTACGACATGAACACCGGCACCCTGAGGAACTACGACCCCAGGATCAACCTGGTGCCCGTGAACAGGCGGCTGCCCCACGCCCTGGTGCTGCACCACAACGAGCACCCACAGAGCGACTTCAGCTCCTTCGTGAGCAAGCTGAAAGGCAGGACCGTGCTGGTCGTGGGCGAGAAGCTGAGCGTGCCCGGCAAGATGGTGGACTGGCTGAGCGACAGGCCCGAGGCCACCTTCCGGGCCAGGCTGGACCTCGGCATCCCCGGCGACGTGCCCAAGTACGACATCATCTTCGTGAACGTCAGGACCCCATACAAGTACCACCATTACCAGCAGTGCGAGGACCACGCCATCAAGCTGAGCATGCTGACCAAGAAGGCCTGCCTGCACCTGAACCCCGGAGGCACCTGCGTGAGCATCGGCTACGGCTACGCCGACAGGGCCAGCGAGAGCATCATTGGCGCCATCGCCAGGCTGTTCAAGTTCAGCAGGGTGTGCAAACCCAAGAGCAGCCTGGAGGAAACCGAGGTGCTGTTCGTGTTCATCGGCTACGACCGGAAGGCCAGGACCCACAACCCCTACAAGCTGAGCAGCACCCTGACAAACATCTACACCGGCAGCAGGCTGCACGAGGCCGGCTGCGCCCCCAGCTACCACGTGGTCAGGGGCGATATCGCCACCGCCACCGAGGGCGTGATCATCAACGCTGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGTGTGCGGCGCCCTGTACAAGAAGTTCCCCGAGAGCTTCGACCTGCAGCCCATCGAGGTGGGCAAGGCCAGGCTGGTGAAGGGCGCCGCTAAGCACATCATCCACGCCGTGGGCCCCAACTTCAACAAGGTGAGCGAGGTGGAAGGCGACAAGCAGCTGGCCGAAGCCTACGAGAGCATCGCCAAGATCGTGAACGACAATAACTACAAGAGCGTGGCCATCCCACTGCTCAGCACCGGCATCTTCAGCGGCAACAAGGACAGGCTGACCCAGAGCCTGAACCACCTGCTCACCGCCCTGGACACCACCGATGCCGACGTGGCCATCTACTGCAGGGACAAGAAGTGGGAGATGACCCTGAAGGAGGCCGTGGCCAGGCGGGAGGCCGTGGAAGAGATCTGCATCAGCGACGACTCCAGCGTGACCGAGCCCGACGCCGAGCTGGTGAGGGTGCACCCCAAGAGCTCCCTGGCCGGCAGGAAGGGCTACAGCACCAGCGACGGCAAGACCTTCAGCTACCTGGAGGGCACCAAGTTCCACCAGGCCGCTAAGGACATCGCCGAGATCAACGCTATGTGGCCCGTGGCCACCGAGGCCAACGAGCAGGTGTGCATGTACATCCTGGGCGAGAGCATGTCCAGCATCAGGAGCAAGTGCCCCGTGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCTGCCCTGCCTGTGCATCCACGCTATGACACCCGAGAGGGTGCAGCGGCTGAAGGCCAGCAGGCCCGAGCAGATCACCGTGTGCAGCTCCTTCCCACTGCCCAAGTACAGGATCACCGGCGTGCAGAAGATCCAGTGCAGCCAGCCCATCCTGTTCAGCCCAAAGGTGCCCGCCTACATCCACCCCAGGAAGTACCTGGTGGAGACCCCACCCGTGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCTGATCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCATCATTATCGAGGAAGAGGAAGAGGACAGCATCAGCCTGCTGAGCGACGGCCCCACCCACCAGGTGCTGCAGGTGGAGGCCGACATCCACGGCCCACCCAGCGTGTCCAGCTCCAGCTGGAGCATCCCACACGCCAGCGACTTCGACGTGGACAGCCTGAGCATCCTGGACACCCTGGAGGGCGCCAGCGTGACCTCCGGCGCCACCAGCGCCGAGACCAACAGCTACTTCGCCAAGAGCATGGAGTTCCTGGCCAGGCCCGTGCCAGCTCCCAGGACCGTGTTCAGGAACCCACCCCACCCAGCTCCCAGGACCAGGACCCCAAGCCTGGCTCCCAGCAGGGCCTGCAGCAGGACCAGCCTGGTGAGCACCCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAACTGGAGGCCCTGACACCCAGCAGGACCCCCAGCAGGTCCGTGAGCAGGACTAGTCTGGTGTCCAACCCACCCGGCGTGAACAGGGTGATCACCAGGGAGGAATTCGAGGCCTTCGTGGCCCAGCAACAGAGACGGTTCGACGCCGGCGCCTACATCTTCAGCAGCGACACCGGCCAGGGACACCTGCAGCAAAAGAGCGTGAGGCAGACCGTGCTGAGCGAGGTGGTGCTGGAGAGGACCGAGCTGGAAATCAGCTACGCCCCCAGGCTGGACCAGGAGAAGGAGGAACTGCTCAGGAAGAAACTGCAGCTGAACCCCACCCCAGCCAACAGGAGCAGGTACCAGAGCAGGAAGGTGGAGAACATGAAGGCCATCACCGCCAGGCGGATCCTGCAGGGCCTGGGACACTACCTGAAGGCCGAGGGCAAGGTGGAGTGCTACAGGACCCTGCACCCCGTGCCACTGTACAGCTCCAGCGTGAACAGGGCCTTCTCCAGCCCCAAGGTGGCCGTGGAGGCCTGCAACGCTATGCTGAAGGAGAACTTCCCCACCGTGGCCAGCTACTGCATCATCCCCGAGTACGACGCCTACCTGGACATGGTGGACGGCGCCAGCTGCTGCCTGGACACCGCCAGCTTCTGCCCCGCCAAGCTGAGGAGCTTCCCCAAGAAACACAGCTACCTGGAGCCCACCATCAGGAGCGCCGTGCCCAGCGCCATCCAGAACACCCTGCAGAACGTGCTGGCCGCTGCCACCAAGAGGAACTGCAACGTGACCCAGATGAGGGAGCTGCCCGTGCTGGACAGCGCTGCCTTCAACGTGGAGTGCTTCAAGAAATACGCCTGCAACAACGAGTACTGGGAGACCTTCAAGGAGAACCCCATCAGGCTGACCGAAGAGAACGTGGTGAACTACATCACCAAGCTGAAGGGCCCCAAGGCCGCTGCCCTGTTCGCTAAGACCCACAACCTGAACATGCTGCAGGACATCCCAATGGACAGGTTCGTGATGGACCTGAAGAGGGACGTGAAGGTGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGTGCAGGTGATCCAGGCCGCTGACCCACTGGCCACCGCCTACCTGTGCGGCATCCACAGGGAGCTGGTGAGGCGGCTGAACGCCGTGCTGCTGCCCAACATCCACACCCTGTTCGACATGAGCGCCGAGGACTTCGACGCCATCATCGCCGAGCACTTCCAGCCCGGCGACTGCGTGCTGGAGACCGACATCGCCAGCTTCGACAAGAGCGAGGATGACGCTATGGCCCTGACCGCTCTGATGATCCTGGAGGACCTGGGCGTGGACGCCGAGCTGCTCACCCTGATCGAGGCTGCCTTCGGCGAGATCAGCTCCATCCACCTGCCCACCAAGACCAAGTTCAAGTTCGGCGCTATGATGAAAAGCGGAATGTTCCTGACCCTGTTCGTGAACACCGTGATCAACATTGTGATCGCCAGCAGGGTGCTGCGGGAGAGGCTGACCGGCAGCCCCTGCGCTGCCTTCATCGGCGACGACAACATCGTGAAGGGCGTGAAAAGCGACAAGCTGATGGCCGACAGGTGCGCCACCTGGCTGAACATGGAGGTGAAGATCATCGACGCCGTGGTGGGCGAGAAGGCCCCCTACTTCTGCGGCGGATTCATCCTGTGCGACAGCGTGACCGGCACCGCCTGCAGGGTGGCCGACCCCCTGAAGAGGCTGTTCAAGCTGGGCAAGCCACTGGCCGCTGACGATGAGCACGACGATGACAGGCGGAGGGCCCTGCACGAGGAAAGCACCAGGTGGAACAGGGTGGGCATCCTGAGCGAGCTGTGCAAGGCCGTGGAGAGCAGGTACGAGACCGTGGGCACCAGCATCATCGTGATGGCTATGACCACACTGGCCAGCTCCGTCAAGAGCTTCTCCTACCTGAGGGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAgccaccATGagcaagatctacatcgacgagcggagcaacgccgagatcgtgtgcgaggccatcaagaccatcggcatcgagggogccaccgccgcccagctgaccaggcagctgaacatggagaagcgggaggtgaacaaggccctgtacgacctgcagaggagcgctatggtgtactccagcgacgacatccctccccggtggttcatgaccaccgaggccgacaagcccgacgccgacgctatggccgacgtgatcatcgacgacgtgagcagggagaagtccatgagggaggaccacaagagcttcgacgacgtgatccccgccaagaagatcatcgactggaagggcgccaaccccgtgaccgtgatcaacgagtactgccagatcaccaggagggactggagcttccggatcgagagcgtgggccccagcaacagccccaccttctacgcctgcgtggacatcgacggcagggtgttcgacaaggccgacggcaagagcaagcgggacgccaagaacaacgccgccaagctggccgtggacaagctgctgggctacgtgatcatccggttcTAAactcgagctagtgactgactaggatctggttaccactaaaccagcctcaagaacacccgaatggagtctctaagctacataataccaacttacacttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaataaaaagaaagtttcttcacattctagAGCTCCGTCAAGAGCTTCTCCTACCTGAGGGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGCCACCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGACAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGTCCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAACTCGAGTATGTTACGTGCAAAGGTGATTGTCACCCCCCGAAAGACCATATTGTGACACACCCTCAGTATCACGCCCAAACATTTACAGCCGCGGTGTCAAAAACCGCGTGGACGTGGTTAACATCCCTGCTGGGAGGATCAGCCGTAATTATTATAATTGGCTTGGTGCTGGCTACTATTGTGGCCATGTACGTGCTGACCAACCAGAAACATAATTGAATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAATctagAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAaaaaaaaaaaaaaaaaaaaa

(RNA sequence for STARR Fluc IRES-E3L) SEQ ID NO:128 AUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAAAUGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGAGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCCGUCGACGGCCCCACCAGCCUGUACCACCAGGCCAACAAGGGCGUGAGGGUGGCCUACUGGAUCGGCUUCGACACCACACCCUUCAUGUUCAAGAACCUGGCCGGCGCCUACCCCAGCUACAGCACCAACUGGGCCGACGAGACCGUGCUGACCGCCAGGAACAUCGGCCUGUGCAGCAGCGACGUGAUGGAGAGGAGCCGGAGAGGCAUGAGCAUCCUGAGGAAGAAAUACCUGAAGCCCAGCAACAACGUGCUGUUCAGCGUGGGCAGCACCAUCUACCACGAGAAGAGGGACCUGCUCAGGAGCUGGCACCUGCCCAGCGUGUUCCACCUGAGGGGCAAGCAGAACUACACCUGCAGGUGCGAGACCAUCGUGAGCUGCGACGGCUACGUGGUGAAGAGGAUCGCCAUCAGCCCCGGCCUGUACGGCAAGCCCAGCGGCUACGCCGCUACAAUGCACAGGGAGGGCUUCCUGUGCUGCAAGGUGACCGACACCCUGAACGGCGAGAGGGUGAGCUUCCCCGUGUGCACCUACGUGCCCGCCACCCUGUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGAGCGCCGACGACGCCCAGAAGCUGCUCGUGGGCCUGAACCAGAGGAUCGUGGUCAACGGCAGGACCCAGAGGAACACCAACACAAUGAAGAACUACCUGCUGCCCGUGGUGGCCCAGGCUUUCGCCAGGUGGGCCAAGGAGUACAAGGAGGACCAGGAAGACGAGAGGCCCCUGGGCCUGAGGGACAGGCAGCUGGUGAUGGGCUGCUGCUGGGCCUUCAGGCGGCACAAGAUCACCAGCAUCUACAAGAGGCCCGACACCCAGACCAUCAUCAAGGUGAACAGCGACUUCCACAGCUUCGUGCUGCCCAGGAUCGGCAGCAACACCCUGGAGAUCGGCCUGAGGACCCGGAUCAGGAAGAUGCUGGAGGAACACAAGGAGCCCAGCCCACUGAUCACCGCCGAGGACGUGCAGGAGGCCAAGUGCGCUGCCGACGAGGCCAAGGAGGUGAGGGAGGCCGAGGAACUGAGGGCCGCCCUGCCACCCCUGGCUGCCGACGUGGAGGAACCCACCCUGGAAGCCGACGUGGACCUGAUGCUGCAGGAGGCCGGCGCCGGAAGCGUGGAGACACCCAGGGGCCUGAUCAAGGUGACCAGCUACGACGGCGAGGACAAGAUCGGCAGCUACGCCGUGCUGAGCCCACAGGCCGUGCUGAAGUCCGAGAAGCUGAGCUGCAUCCACCCACUGGCCGAGCAGGUGAUCGUGAUCACCCACAGCGGCAGGAAGGGCAGGUACGCCGUGGAGCCCUACCACGGCAAGGUGGUCGUGCCCGAGGGCCACGCCAUCCCCGUGCAGGACUUCCAGGCCCUGAGCGAGAGCGCCACCAUCGUGUACAACGAGAGGGAGUUCGUGAACAGGUACCUGCACCAUAUCGCCACCCACGGCGGAGCCCUGAACACCGACGAGGAAUACUACAAGACCGUGAAGCCCAGCGAGCACGACGGCGAGUACCUGUACGACAUCGACAGGAAGCAGUGCGUGAAGAAAGAGCUGGUGACCGGCCUGGGACUGACCGGCGAGCUGGUGGACCCACCCUUCCACGAGUUCGCCUACGAGAGCCUGAGGACCAGACCCGCCGCUCCCUACCAGGUGCCCACCAUCGGCGUGUACGGCGUGCCCGGCAGCGGAAAGAGCGGCAUCAUCAAGAGCGCCGUGACCAAGAAAGACCUGGUGGUCAGCGCCAAGAAAGAGAACUGCGCCGAGAUCAUCAGGGACGUGAAGAAGAUGAAAGGCCUGGACGUGAACGCGCGCACCGUGGACAGCGUGCUGCUGAACGGCUGCAAGCACCCCGUGGAGACCCUGUACAUCGACGAGGCCUUCGCUUGCCACGCCGGCACCCUGAGGGCCCUGAUCGCCAUCAUCAGGCCCAAGAAAGCCGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUGAUGUGCCUGAAGGUGCACUUCAACCACGAGAUCUGCACCCAGGUGUUCCACAAGAGCAUCAGCAGGCGGUGCACCAAGAGCGUGACCAGCGUCGUGAGCACCCUGUUCUACGACAAGAAAAUGAGGACCACCAACCCCAAGGAGACCAAAAUCGUGAUCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCUGAUCCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCAGAUCGACUACAAGGGCAACGAGAUCAUGACCGCCGCUGCCAGCCAGGGCCUGACCAGGAAGGGCGUGUACGCCGUGAGGUACAAGGUGAACGAGAACCCACUGUACGCUCCCACCAGCGAGCACGUGAACGUGCUGCUGACCAGGACCGAGGACAGGAUCGUGUGGAAGACCCUGGCCGGCGACCCCUGGAUCAAGACCCUGACCGCCAAGUACCCCGGCAACUUCACCGCCACCAUCGAAGAGUGGCAGGCCGAGCACGACGCCAUCAUGAGGCACAUCCUGGAGAGGCCCGACCCCACCGACGUGUUCCAGAACAAGGCCAACGUGUGCUGGGCCAAGGCCCUGGUGCCCGUGCUGAAGACCGCCGGCAUCGACAUGACCACAGAGCAGUGGAACACCGUGGACUACUUCGAGACCGACAAGGCCCACAGCGCCGAGAUCGUGCUGAACCAGCUGUGCGUGAGGUUCUUCGGCCUGGACCUGGACAGCGGCCUGUUCAGCGCCCCCACCGUGCCACUGAGCAUCAGGAACAACCACUGGGACAACAGCCCCAGCCCAAACAUGUACGGCCUGAACAAGGAGGUGGUCAGGCAGCUGAGCAGGCGGUACCCACAGCUGCCCAGGGCCGUGGCCACCGGCAGGGUGUACGACAUGAACACCGGCACCCUGAGGAACUACGACCCCAGGAUCAACCUGGUGCCCGUGAACAGGCGGCUGCCCCACGCCCUGGUGCUGCACCACAACGAGCACCCACAGAGCGACUUCAGCUCCUUCGUGAGCAAGCUGAAAGGCAGGACCGUGCUGGUCGUGGGCGAGAAGCUGAGCGUGCCCGGCAAGAUGGUGGACUGGCUGAGCGACAGGCCCGAGGCCACCUUCCGGGCCAGGCUGGACCUCGGCAUCCCCGGCGACGUGCCCAAGUACGACAUCAUCUUCGUGAACGUCAGGACCCCAUACAAGUACCACCAUUACCAGCAGUGCGAGGACCACGCCAUCAAGCUGAGCAUGCUGACCAAGAAGGCCUGCCUGCACCUGAACCCCGGAGGCACCUGCGUGAGCAUCGGCUACGGCUACGCCGACAGGGCCAGCGAGAGCAUCAUUGGCGCCAUCGCCAGGCUGUUCAAGUUCAGCAGGGUGUGCAAACCCAAGAGCAGCCUGGAGGAAACCGAGGUGCUGUUCGUGUUCAUCGGCUACGACCGGAAGGCCAGGACCCACAACCCCUACAAGCUGAGCAGCACCCUGACAAACAUCUACACCGGCAGCAGGCUGCACGAGGCCGGCUGCGCCCCCAGCUACCACGUGGUCAGGGGCGAUAUCGCCACCGCCACCGAGGGCGUGAUCAUCAACGCUGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGUGUGCGGCGCCCUGUACAAGAAGUUCCCCGAGAGCUUCGACCUGCAGCCCAUCGAGGUGGGCAAGGCCAGGCUGGUGAAGGGCGCCGCUAAGCACAUCAUCCACGCCGUGGGCCCCAACUUCAACAAGGUGAGCGAGGUGGAAGGCGACAAGCAGCUGGCCGAAGCCUACGAGAGCAUCGCCAAGAUCGUGAACGACAAUAACUACAAGAGCGUGGCCAUCCCACUGCUCAGCACCGGCAUCUUCAGCGGCAACAAGGACAGGCUGACCCAGAGCCUGAACCACCUGCUCACCGCCCUGGACACCACCGAUGCCGACGUGGCCAUCUACUGCAGGGACAAGAAGUGGGAGAUGACCCUGAAGGAGGCCGUGGCCAGGCGGGAGGCCGUGGAAGAGAUCUGCAUCAGCGACGACUCCAGCGUGACCGAGCCCGACGCCGAGCUGGUGAGGGUGCACCCCAAGAGCUCCCUGGCCGGCAGGAAGGGCUACAGCACCAGCGACGGCAAGACCUUCAGCUACCUGGAGGGCACCAAGUUCCACCAGGCCGCUAAGGACAUCGCCGAGAUCAACGCUAUGUGGCCCGUGGCCACCGAGGCCAACGAGCAGGUGUGCAUGUACAUCCUGGGCGAGAGCAUGUCCAGCAUCAGGAGCAAGUGCCCCGUGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCUGCCCUGCCUGUGCAUCCACGCUAUGACACCCGAGAGGGUGCAGCGGCUGAAGGCCAGCAGGCCCGAGCAGAUCACCGUGUGCAGCUCCUUCCCACUGCCCAAGUACAGGAUCACCGGCGUGCAGAAGAUCCAGUGCAGCCAGCCCAUCCUGUUCAGCCCAAAGGUGCCCGCCUACAUCCACCCCAGGAAGUACCUGGUGGAGACCCCACCCGUGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCUGAUCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCAUCAUUAUCGAGGAAGAGGAAGAGGACAGCAUCAGCCUGCUGAGCGACGGCCCCACCCACCAGGUGCUGCAGGUGGAGGCCGACAUCCACGGCCCACCCAGCGUGUCCAGCUCCAGCUGGAGCAUCCCACACGCCAGCGACUUCGACGUGGACAGCCUGAGCAUCCUGGACACCCUGGAGGGCGCCAGCGUGACCUCCGGCGCCACCAGCGCCGAGACCAACAGCUACUUCGCCAAGAGCAUGGAGUUCCUGGCCAGGCCCGUGCCAGCUCCCAGGACCGUGUUCAGGAACCCACCCCACCCAGCUCCCAGGACCAGGACCCCAAGCCUGGCUCCCAGCAGGGCCUGCAGCAGGACCAGCCUGGUGAGCACCCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAACUGGAGGCCCUGACACCCAGCAGGACCCCCAGCAGGUCCGUGAGCAGGACUAGUCUGGUGUCCAACCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAAUUCGAGGCCUUCGUGGCCCAGCAACAGAGACGGUUCGACGCCGGCGCCUACAUCUUCAGCAGCGACACCGGCCAGGGACACCUGCAGCAAAAGAGCGUGAGGCAGACCGUGCUGAGCGAGGUGGUGCUGGAGAGGACCGAGCUGGAAAUCAGCUACGCCCCCAGGCUGGACCAGGAGAAGGAGGAACUGCUCAGGAAGAAACUGCAGCUGAACCCCACCCCAGCCAACAGGAGCAGGUACCAGAGCAGGAAGGUGGAGAACAUGAAGGCCAUCACCGCCAGGCGGAUCCUGCAGGGCCUGGGACACUACCUGAAGGCCGAGGGCAAGGUGGAGUGCUACAGGACCCUGCACCCCGUGCCACUGUACAGCUCCAGCGUGAACAGGGCCUUCUCCAGCCCCAAGGUGGCCGUGGAGGCCUGCAACGCUAUGCUGAAGGAGAACUUCCCCACCGUGGCCAGCUACUGCAUCAUCCCCGAGUACGACGCCUACCUGGACAUGGUGGACGGCGCCAGCUGCUGCCUGGACACCGCCAGCUUCUGCCCCGCCAAGCUGAGGAGCUUCCCCAAGAAACACAGCUACCUGGAGCCCACCAUCAGGAGCGCCGUGCCCAGCGCCAUCCAGAACACCCUGCAGAACGUGCUGGCCGCUGCCACCAAGAGGAACUGCAACGUGACCCAGAUGAGGGAGCUGCCCGUGCUGGACAGCGCUGCCUUCAACGUGGAGUGCUUCAAGAAAUACGCCUGCAACAACGAGUACUGGGAGACCUUCAAGGAGAACCCCAUCAGGCUGACCGAAGAGAACGUGGUGAACUACAUCACCAAGCUGAAGGGCCCCAAGGCCGCUGCCCUGUUCGCUAAGACCCACAACCUGAACAUGCUGCAGGACAUCCCAAUGGACAGGUUCGUGAUGGACCUGAAGAGGGACGUGAAGGUGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGUGCAGGUGAUCCAGGCCGCUGACCCACUGGCCACCGCCUACCUGUGCGGCAUCCACAGGGAGCUGGUGAGGCGGCUGAACGCCGUGCUGCUGCCCAACAUCCACACCCUGUUCGACAUGAGCGCCGAGGACUUCGACGCCAUCAUCGCCGAGCACUUCCAGCCCGGCGACUGCGUGCUGGAGACCGACAUCGCCAGCUUCGACAAGAGCGAGGAUGACGCUAUGGCCCUGACCGCUCUGAUGAUCCUGGAGGACCUGGGCGUGGACGCCGAGCUGCUCACCCUGAUCGAGGCUGCCUUCGGCGAGAUCAGCUCCAUCCACCUGCCCACCAAGACCAAGUUCAAGUUCGGCGCUAUGAUGAAAAGCGGAAUGUUCCUGACCCUGUUCGUGAACACCGUGAUCAACAUUGUGAUCGCCAGCAGGGUGCUGCGGGAGAGGCUGACCGGCAGCCCCUGCGCUGCCUUCAUCGGCGACGACAACAUCGUGAAGGGCGUGAAAAGCGACAAGCUGAUGGCCGACAGGUGCGCCACCUGGCUGAACAUGGAGGUGAAGAUCAUCGACGCCGUGGUGGGCGAGAAGGCCCCCUACUUCUGCGGCGGAUUCAUCCUGUGCGACAGCGUGACCGGCACCGCCUGCAGGGUGGCCGACCCCCUGAAGAGGCUGUUCAAGCUGGGCAAGCCACUGGCCGCUGACGAUGAGCACGACGAUGACAGGCGGAGGGCCCUGCACGAGGAAAGCACCAGGUGGAACAGGGUGGGCAUCCUGAGCGAGCUGUGCAAGGCCGUGGAGAGCAGGUACGAGACCGUGGGCACCAGCAUCAUCGUGAUGGCUAUGACCACACUGGCCAGCUCCGUCAAGAGCUUCUCCUACCUGAGGGGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGGCCGCCACCAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGACAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGUCCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAACUCGAGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACAUUAAAACAGCCUGUGGGUUGAUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGGUAUCACGGUACCUUUGUGCGCCUGUUUUAUACCCCCUCCCCCAACUGUAACUUAGAAGUAACACACACCGAUCAACAGUCAGCGUGGCACACCAGCCACGUUUUGAUCAAGCACUUCUGUUACCCCGGACUGAGUAUCAAUAGACUGCUCACGCGGUUGAAGGAGAAAGCGUUCGUUAUCCGGCCAACUACUUCGAAAAACCUAGUAACACCGUGGAAGUUGCAGAGUGUUUCGCUCAGCACUACCCCAGUGUAGAUCAGGUCGAUGAGUCACCGCAUUCCCCACGGGCGACCGUGGCGGUGGCUGCGUUGGCGGCCUGCCCAUGGGGAAACCCAUGGGACGCUCUAAUACAGACAUGGUGCGAAGAGUCUAUUGAGCUAGUUGGUAGUCCUCCGGCCCCUGAAUGCGGCUAAUCCUAACUGCGGAGCACACACCCUCAAGCCAGAGGGCAGUGUGUCGUAACGGGCAACUCUGCAGCGGAACCGACUACUUUGGGUGUCCGUGUUUCAUUUUAUUCCUAUACUGGCUGCUUAUGGUGACAAUUGAGAGAUCGUUACCAUAUAGCUAUUGGAUUGGCCAUCCGGUGACUAAUAGAGCUAUUAUAUAUCCCUUUGUUGGGUUUAUACCACUUAGCUUGAAAGAGGUUAAAACAUUACAAUUCAUUGUUAAGUUGAAUACAGCAAAAUGAGCAAGAUCUACAUCGACGAGCGGAGCAACGCCGAGAUCGUGUGCGAGGCCAUCAAGACCAUCGGCAUCGAGGGCGCCACCGCCGCCCAGCUGACCAGGCAGCUGAACAUGGAGAAGCGGGAGGUGAACAAGGCCCUGUACGACCUGCAGAGGAGCGCUAUGGUGUACUCCAGCGACGACAUCCCUCCCCGGUGGUUCAUGACCACCGAGGCCGACAAGCCCGACGCCGACGCUAUGGCCGACGUGAUCAUCGACGACGUGAGCAGGGAGAAGUCCAUGAGGGAGGACCACAAGAGCUUCGACGACGUGAUCCCCGCCAAGAAGAUCAUCGACUGGAAGGGCGCCAACCCCGUGACCGUGAUCAACGAGUACUGCCAGAUCACCAGGAGGGACUGGAGCUUCCGGAUCGAGAGCGUGGGCCCCAGCAACAGCCCCACCUUCUACGCCUGCGUGGACAUCGACGGCAGGGUGUUCGACAAGGCCGACGGCAAGAGCAAGCGGGACGCCAAGAACAACGCCGCCAAGCUGGCCGUGGACAAGCUGCUGGGCUACGUGAUCAUCCGGUUCUAAACGUAUGUUACGUGCAAAGGUGAUUGUCACCCCCCGAAAGACCAUAUUGUGACACACCCUCAGUAUCACGCCCAAACAUUUACAGCCGCGGUGUCAAAAACCGCGUGGACGUGGUUAACAUCCCUGCUGGGAGGAUCAGCCGUAAUUAUUAUAAUUGGCUUGGUGCUGGCUACUAUUGUGGCCAUGUACGUGCUGACCAACCAGAAACAUAAUUGAAUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

(RNA sequence for STARR Fluc IRES-E3L (short 3′ UTR)) SEQ ID NO:129 AUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAAAUGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGAGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCCGUCGACGGCCCCACCAGCCUGUACCACCAGGCCAACAAGGGCGUGAGGGUGGCCUACUGGAUCGGCUUCGACACCACACCCUUCAUGUUCAAGAACCUGGCCGGCGCCUACCCCAGCUACAGCACCAACUGGGCCGACGAGACCGUGCUGACCGCCAGGAACAUCGGCCUGUGCAGCAGCGACGUGAUGGAGAGGAGCCGGAGAGGCAUGAGCAUCCUGAGGAAGAAAUACCUGAAGCCCAGCAACAACGUGCUGUUCAGCGUGGGCAGCACCAUCUACCACGAGAAGAGGGACCUGCUCAGGAGCUGGCACCUGCCCAGCGUGUUCCACCUGAGGGGCAAGCAGAACUACACCUGCAGGUGCGAGACCAUCGUGAGCUGCGACGGCUACGUGGUGAAGAGGAUCGCCAUCAGCCCCGGCCUGUACGGCAAGCCCAGCGGCUACGCCGCUACAAUGCACAGGGAGGGCUUCCUGUGCUGCAAGGUGACCGACACCCUGAACGGCGAGAGGGUGAGCUUCCCCGUGUGCACCUACGUGCCCGCCACCCUGUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGAGCGCCGACGACGCCCAGAAGCUGCUCGUGGGCCUGAACCAGAGGAUCGUGGUCAACGGCAGGACCCAGAGGAACACCAACACAAUGAAGAACUACCUGCUGCCCGUGGUGGCCCAGGCUUUCGCCAGGUGGGCCAAGGAGUACAAGGAGGACCAGGAAGACGAGAGGCCCCUGGGCCUGAGGGACAGGCAGCUGGUGAUGGGCUGCUGCUGGGCCUUCAGGCGGCACAAGAUCACCAGCAUCUACAAGAGGCCCGACACCCAGACCAUCAUCAAGGUGAACAGCGACUUCCACAGCUUCGUGCUGCCCAGGAUCGGCAGCAACACCCUGGAGAUCGGCCUGAGGACCCGGAUCAGGAAGAUGCUGGAGGAACACAAGGAGCCCAGCCCACUGAUCACCGCCGAGGACGUGCAGGAGGCCAAGUGCGCUGCCGACGAGGCCAAGGAGGUGAGGGAGGCCGAGGAACUGAGGGCCGCCCUGCCACCCCUGGCUGCCGACGUGGAGGAACCCACCCUGGAAGCCGACGUGGACCUGAUGCUGCAGGAGGCCGGCGCCGGAAGCGUGGAGACACCCAGGGGCCUGAUCAAGGUGACCAGCUACGACGGCGAGGACAAGAUCGGCAGCUACGCCGUGCUGAGCCCACAGGCCGUGCUGAAGUCCGAGAAGCUGAGCUGCAUCCACCCACUGGCCGAGCAGGUGAUCGUGAUCACCCACAGCGGCAGGAAGGGCAGGUACGCCGUGGAGCCCUACCACGGCAAGGUGGUCGUGCCCGAGGGCCACGCCAUCCCCGUGCAGGACUUCCAGGCCCUGAGCGAGAGCGCCACCAUCGUGUACAACGAGAGGGAGUUCGUGAACAGGUACCUGCACCAUAUCGCCACCCACGGCGGAGCCCUGAACACCGACGAGGAAUACUACAAGACCGUGAAGCCCAGCGAGCACGACGGCGAGUACCUGUACGACAUCGACAGGAAGCAGUGCGUGAAGAAAGAGCUGGUGACCGGCCUGGGACUGACCGGCGAGCUGGUGGACCCACCCUUCCACGAGUUCGCCUACGAGAGCCUGAGGACCAGACCCGCCGCUCCCUACCAGGUGCCCACCAUCGGCGUGUACGGCGUGCCCGGCAGCGGAAAGAGCGGCAUCAUCAAGAGCGCCGUGACCAAGAAAGACCUGGUGGUCAGCGCCAAGAAAGAGAACUGCGCCGAGAUCAUCAGGGACGUGAAGAAGAUGAAAGGCCUGGACGUGAACGCGCGCACCGUGGACAGCGUGCUGCUGAACGGCUGCAAGCACCCCGUGGAGACCCUGUACAUCGACGAGGCCUUCGCUUGCCACGCCGGCACCCUGAGGGCCCUGAUCGCCAUCAUCAGGCCCAAGAAAGCCGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUGAUGUGCCUGAAGGUGCACUUCAACCACGAGAUCUGCACCCAGGUGUUCCACAAGAGCAUCAGCAGGCGGUGCACCAAGAGCGUGACCAGCGUCGUGAGCACCCUGUUCUACGACAAGAAAAUGAGGACCACCAACCCCAAGGAGACCAAAAUCGUGAUCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCUGAUCCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCAGAUCGACUACAAGGGCAACGAGAUCAUGACCGCCGCUGCCAGCCAGGGCCUGACCAGGAAGGGCGUGUACGCCGUGAGGUACAAGGUGAACGAGAACCCACUGUACGCUCCCACCAGCGAGCACGUGAACGUGCUGCUGACCAGGACCGAGGACAGGAUCGUGUGGAAGACCCUGGCCGGCGACCCCUGGAUCAAGACCCUGACCGCCAAGUACCCCGGCAACUUCACCGCCACCAUCGAAGAGUGGCAGGCCGAGCACGACGCCAUCAUGAGGCACAUCCUGGAGAGGCCCGACCCCACCGACGUGUUCCAGAACAAGGCCAACGUGUGCUGGGCCAAGGCCCUGGUGCCCGUGCUGAAGACCGCCGGCAUCGACAUGACCACAGAGCAGUGGAACACCGUGGACUACUUCGAGACCGACAAGGCCCACAGCGCCGAGAUCGUGCUGAACCAGCUGUGCGUGAGGUUCUUCGGCCUGGACCUGGACAGCGGCCUGUUCAGCGCCCCCACCGUGCCACUGAGCAUCAGGAACAACCACUGGGACAACAGCCCCAGCCCAAACAUGUACGGCCUGAACAAGGAGGUGGUCAGGCAGCUGAGCAGGCGGUACCCACAGCUGCCCAGGGCCGUGGCCACCGGCAGGGUGUACGACAUGAACACCGGCACCCUGAGGAACUACGACCCCAGGAUCAACCUGGUGCCCGUGAACAGGCGGCUGCCCCACGCCCUGGUGCUGCACCACAACGAGCACCCACAGAGCGACUUCAGCUCCUUCGUGAGCAAGCUGAAAGGCAGGACCGUGCUGGUCGUGGGCGAGAAGCUGAGCGUGCCCGGCAAGAUGGUGGACUGGCUGAGCGACAGGCCCGAGGCCACCUUCCGGGCCAGGCUGGACCUCGGCAUCCCCGGCGACGUGCCCAAGUACGACAUCAUCUUCGUGAACGUCAGGACCCCAUACAAGUACCACCAUUACCAGCAGUGCGAGGACCACGCCAUCAAGCUGAGCAUGCUGACCAAGAAGGCCUGCCUGCACCUGAACCCCGGAGGCACCUGCGUGAGCAUCGGCUACGGCUACGCCGACAGGGCCAGCGAGAGCAUCAUUGGCGCCAUCGCCAGGCUGUUCAAGUUCAGCAGGGUGUGCAAACCCAAGAGCAGCCUGGAGGAAACCGAGGUGCUGUUCGUGUUCAUCGGCUACGACCGGAAGGCCAGGACCCACAACCCCUACAAGCUGAGCAGCACCCUGACAAACAUCUACACCGGCAGCAGGCUGCACGAGGCCGGCUGCGCCCCCAGCUACCACGUGGUCAGGGGCGAUAUCGCCACCGCCACCGAGGGCGUGAUCAUCAACGCUGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGUGUGCGGCGCCCUGUACAAGAAGUUCCCCGAGAGCUUCGACCUGCAGCCCAUCGAGGUGGGCAAGGCCAGGCUGGUGAAGGGCGCCGCUAAGCACAUCAUCCACGCCGUGGGCCCCAACUUCAACAAGGUGAGCGAGGUGGAAGGCGACAAGCAGCUGGCCGAAGCCUACGAGAGCAUCGCCAAGAUCGUGAACGACAAUAACUACAAGAGCGUGGCCAUCCCACUGCUCAGCACCGGCAUCUUCAGCGGCAACAAGGACAGGCUGACCCAGAGCCUGAACCACCUGCUCACCGCCCUGGACACCACCGAUGCCGACGUGGCCAUCUACUGCAGGGACAAGAAGUGGGAGAUGACCCUGAAGGAGGCCGUGGCCAGGCGGGAGGCCGUGGAAGAGAUCUGCAUCAGCGACGACUCCAGCGUGACCGAGCCCGACGCCGAGCUGGUGAGGGUGCACCCCAAGAGCUCCCUGGCCGGCAGGAAGGGCUACAGCACCAGCGACGGCAAGACCUUCAGCUACCUGGAGGGCACCAAGUUCCACCAGGCCGCUAAGGACAUCGCCGAGAUCAACGCUAUGUGGCCCGUGGCCACCGAGGCCAACGAGCAGGUGUGCAUGUACAUCCUGGGCGAGAGCAUGUCCAGCAUCAGGAGCAAGUGCCCCGUGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCUGCCCUGCCUGUGCAUCCACGCUAUGACACCCGAGAGGGUGCAGCGGCUGAAGGCCAGCAGGCCCGAGCAGAUCACCGUGUGCAGCUCCUUCCCACUGCCCAAGUACAGGAUCACCGGCGUGCAGAAGAUCCAGUGCAGCCAGCCCAUCCUGUUCAGCCCAAAGGUGCCCGCCUACAUCCACCCCAGGAAGUACCUGGUGGAGACCCCACCCGUGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCUGAUCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCAUCAUUAUCGAGGAAGAGGAAGAGGACAGCAUCAGCCUGCUGAGCGACGGCCCCACCCACCAGGUGCUGCAGGUGGAGGCCGACAUCCACGGCCCACCCAGCGUGUCCAGCUCCAGCUGGAGCAUCCCACACGCCAGCGACUUCGACGUGGACAGCCUGAGCAUCCUGGACACCCUGGAGGGCGCCAGCGUGACCUCCGGCGCCACCAGCGCCGAGACCAACAGCUACUUCGCCAAGAGCAUGGAGUUCCUGGCCAGGCCCGUGCCAGCUCCCAGGACCGUGUUCAGGAACCCACCCCACCCAGCUCCCAGGACCAGGACCCCAAGCCUGGCUCCCAGCAGGGCCUGCAGCAGGACCAGCCUGGUGAGCACCCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAACUGGAGGCCCUGACACCCAGCAGGACCCCCAGCAGGUCCGUGAGCAGGACUAGUCUGGUGUCCAACCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAAUUCGAGGCCUUCGUGGCCCAGCAACAGAGACGGUUCGACGCCGGCGCCUACAUCUUCAGCAGCGACACCGGCCAGGGACACCUGCAGCAAAAGAGCGUGAGGCAGACCGUGCUGAGCGAGGUGGUGCUGGAGAGGACCGAGCUGGAAAUCAGCUACGCCCCCAGGCUGGACCAGGAGAAGGAGGAACUGCUCAGGAAGAAACUGCAGCUGAACCCCACCCCAGCCAACAGGAGCAGGUACCAGAGCAGGAAGGUGGAGAACAUGAAGGCCAUCACCGCCAGGCGGAUCCUGCAGGGCCUGGGACACUACCUGAAGGCCGAGGGCAAGGUGGAGUGCUACAGGACCCUGCACCCCGUGCCACUGUACAGCUCCAGCGUGAACAGGGCCUUCUCCAGCCCCAAGGUGGCCGUGGAGGCCUGCAACGCUAUGCUGAAGGAGAACUUCCCCACCGUGGCCAGCUACUGCAUCAUCCCCGAGUACGACGCCUACCUGGACAUGGUGGACGGCGCCAGCUGCUGCCUGGACACCGCCAGCUUCUGCCCCGCCAAGCUGAGGAGCUUCCCCAAGAAACACAGCUACCUGGAGCCCACCAUCAGGAGCGCCGUGCCCAGCGCCAUCCAGAACACCCUGCAGAACGUGCUGGCCGCUGCCACCAAGAGGAACUGCAACGUGACCCAGAUGAGGGAGCUGCCCGUGCUGGACAGCGCUGCCUUCAACGUGGAGUGCUUCAAGAAAUACGCCUGCAACAACGAGUACUGGGAGACCUUCAAGGAGAACCCCAUCAGGCUGACCGAAGAGAACGUGGUGAACUACAUCACCAAGCUGAAGGGCCCCAAGGCCGCUGCCCUGUUCGCUAAGACCCACAACCUGAACAUGCUGCAGGACAUCCCAAUGGACAGGUUCGUGAUGGACCUGAAGAGGGACGUGAAGGUGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGUGCAGGUGAUCCAGGCCGCUGACCCACUGGCCACCGCCUACCUGUGCGGCAUCCACAGGGAGCUGGUGAGGCGGCUGAACGCCGUGCUGCUGCCCAACAUCCACACCCUGUUCGACAUGAGCGCCGAGGACUUCGACGCCAUCAUCGCCGAGCACUUCCAGCCCGGCGACUGCGUGCUGGAGACCGACAUCGCCAGCUUCGACAAGAGCGAGGAUGACGCUAUGGCCCUGACCGCUCUGAUGAUCCUGGAGGACCUGGGCGUGGACGCCGAGCUGCUCACCCUGAUCGAGGCUGCCUUCGGCGAGAUCAGCUCCAUCCACCUGCCCACCAAGACCAAGUUCAAGUUCGGCGCUAUGAUGAAAAGCGGAAUGUUCCUGACCCUGUUCGUGAACACCGUGAUCAACAUUGUGAUCGCCAGCAGGGUGCUGCGGGAGAGGCUGACCGGCAGCCCCUGCGCUGCCUUCAUCGGCGACGACAACAUCGUGAAGGGCGUGAAAAGCGACAAGCUGAUGGCCGACAGGUGCGCCACCUGGCUGAACAUGGAGGUGAAGAUCAUCGACGCCGUGGUGGGCGAGAAGGCCCCCUACUUCUGCGGCGGAUUCAUCCUGUGCGACAGCGUGACCGGCACCGCCUGCAGGGUGGCCGACCCCCUGAAGAGGCUGUUCAAGCUGGGCAAGCCACUGGCCGCUGACGAUGAGCACGACGAUGACAGGCGGAGGGCCCUGCACGAGGAAAGCACCAGGUGGAACAGGGUGGGCAUCCUGAGCGAGCUGUGCAAGGCCGUGGAGAGCAGGUACGAGACCGUGGGCACCAGCAUCAUCGUGAUGGCUAUGACCACACUGGCCAGCUCCGUCAAGAGCUUCUCCUACCUGAGGGGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGGCCGCCACCAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGACAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGUCCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAACUCGAGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACAUUAAAACAGCCUGUGGGUUGAUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGGUAUCACGGUACCUUUGUGCGCCUGUUUUAUACCCCCUCCCCCAACUGUAACUUAGAAGUAACACACACCGAUCAACAGUCAGCGUGGCACACCAGCCACGUUUUGAUCAAGCACUUCUGUUACCCCGGACUGAGUAUCAAUAGACUGCUCACGCGGUUGAAGGAGAAAGCGUUCGUUAUCCGGCCAACUACUUCGAAAAACCUAGUAACACCGUGGAAGUUGCAGAGUGUUUCGCUCAGCACUACCCCAGUGUAGAUCAGGUCGAUGAGUCACCGCAUUCCCCACGGGCGACCGUGGCGGUGGCUGCGUUGGCGGCCUGCCCAUGGGGAAACCCAUGGGACGCUCUAAUACAGACAUGGUGCGAAGAGUCUAUUGAGCUAGUUGGUAGUCCUCCGGCCCCUGAAUGCGGCUAAUCCUAACUGCGGAGCACACACCCUCAAGCCAGAGGGCAGUGUGUCGUAACGGGCAACUCUGCAGCGGAACCGACUACUUUGGGUGUCCGUGUUUCAUUUUAUUCCUAUACUGGCUGCUUAUGGUGACAAUUGAGAGAUCGUUACCAUAUAGCUAUUGGAUUGGCCAUCCGGUGACUAAUAGAGCUAUUAUAUAUCCCUUUGUUGGGUUUAUACCACUUAGCUUGAAAGAGGUUAAAACAUUACAAUUCAUUGUUAAGUUGAAUACAGCAAAAUGAGCAAGAUCUACAUCGACGAGCGGAGCAACGCCGAGAUCGUGUGCGAGGCCAUCAAGACCAUCGGCAUCGAGGGCGCCACCGCCGCCCAGCUGACCAGGCAGCUGAACAUGGAGAAGCGGGAGGUGAACAAGGCCCUGUACGACCUGCAGAGGAGCGCUAUGGUGUACUCCAGCGACGACAUCCCUCCCCGGUGGUUCAUGACCACCGAGGCCGACAAGCCCGACGCCGACGCUAUGGCCGACGUGAUCAUCGACGACGUGAGCAGGGAGAAGUCCAUGAGGGAGGACCACAAGAGCUUCGACGACGUGAUCCCCGCCAAGAAGAUCAUCGACUGGAAGGGCGCCAACCCCGUGACCGUGAUCAACGAGUACUGCCAGAUCACCAGGAGGGACUGGAGCUUCCGGAUCGAGAGCGUGGGCCCCAGCAACAGCCCCACCUUCUACGCCUGCGUGGACAUCGACGGCAGGGUGUUCGACAAGGCCGACGGCAAGAGCAAGCGGGACGCCAAGAACAACGCCGCCAAGCUGGCCGUGGACAAGCUGCUGGGCUACGUGAUCAUCCGGUUCUAAACAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA

TABLE 8 SEQ ID NO Description SEQ ID NO: 72 nsP1-4 ORF, codon-optimizedSEQ ID NO: 73 5′ UTR SEQ ID NO: 74 5′ UTR SEQ ID NO: 75 5′ UTR SEQ IDNO: 76 3′ UTR SEQ ID NO: 121 SARS-CoV-2 spike glycoprotein (non-codonoptimized nucleic acid) SEQ ID NO: 122 SARS-CoV-2 spike glycoprotein(codon-optimized nucleic acid) SEQ ID NO: 123 SARS-CoV-2 spikeglycoprotein (wild-type protein) SEQ ID NO: 77 Intergenic region betweennsP1-4 ORF and antigenic protein ORF SEQ ID NO: 78 Replicon sequencecomprising SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 76, and SEQ ID NO:77 SEQ ID NO: 124 Replicon sequence comprising SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO: 76, SEQ ID NO: 121, and SEQ ID NO: 77 SEQ ID NO: 125Replicon sequence comprising SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO:76, SEQ ID NO: 122, and SEQ ID NO: 77 (ARCT-021; aka “STARR ™ SARS-CoV-2RNA) SEQ ID NO: 79 nsP1-4 protein sequence SEQ ID NO: 80 nsP1-4 proteinsequence SEQ ID NO: 81 nsP1-4 protein sequence SEQ ID NO: 126 mRNAencoding SARS-CoV-2 glycoprotein SEQ ID NO: 82 5′ UTR (TEV) SEQ ID NO:83 3′ UTR (Xbg)

Example 10

This example describes characterization of self-replicating (STARR™)technology using firefly luciferase transgene expression.

In vitro transcripts were formulated with lipid nanoparticles (LNP) at aconcentration of 0.1 mg/ml, and injected intramuscularly in both legs offemale BALB/C mice (n=3) at a dose of 5 ug per leg. Expression offirefly luciferase (FLuc) was measured by IVIS Lumina LT Series III(PerkinElmer) by administering 100 ul of 1.5 mg Xenolight D-luciferin(PerkinElmer) in PBS via intraperitoneal injection ˜10 min prior to themeasurement. Six data points per group of mice were obtained at eachtime point (FIGS. 18A-18D).

Firefly luciferase (FLuc) expression was monitored from STARR™ Fluc,SINV FLuc, and mRNA FLuc up to day 28 by In Vivo Imaging System (IVIS).Enhanced levels and durations of transgene expression from STARR™ wereobserved. The expression from STARR™ Fluc peaked around day 3 to 7 anddeclined until day 22. Fluc expression from SINV FLuc also peaked on day10, however, the expression was reduced at a significantly faster ratethan STARR™ FLuc. Additionally, the expression on day 3 wassignificantly lower than STARR™ FLuc. FLuc expression from theconventional mRNA backbone was highest at day 1, the earliest time pointin this study, and declined at a slightly faster rate than that ofSTARR™-Fluc (FIG. 18A). FIG. 18B shows that at 14 days post dosing, FLucexpression from STARR™ FLuc was higher than the other groups by abouttwo orders of magnitude. FIG. 18D shows that the effect of the STARR™backbone remained minimal throughout the experimental period (up to day28), while prior administration of SINV replicon backbone resulted in areduction of FLuc transgene expression by ˜2 orders of magnitude.

A cancer vaccine substrate, TA STARR™, was constructed next with theSTARR™ backbone that encodes AH1A5 epitope from gp70, an envelopeglycoprotein of endogenous Murine leukemia virus. AH1 (SPSYVYHQF)(SEQ IDNO:110) is an H-2Ld-restricted antigen of gp70423-431, which isexpressed in tumor cells such as the CT26 colorectal cancer cell line,but not expressed in most of the normal tissues. AH1-A5 is a mutatedsequence with SPSYAYHQF (SEQ ID NO:111) (the mutation underlined) withenhanced affinity to the T cell receptor (Slansky, et al., 2000,Immunity 13: 529-538). The open reading frame of the TA STARR™subgenomic RNA contains a cassette with a signal peptide from the HLAclass I antigen, gp70 sequence containing AH1A5 epitope, ovalbuminepitope (OVA323-339), and MHC class I trafficking signal (Kreiter, etal. 2008, J Immunol 180: 309-318). Three female BALB/c mice wereintramuscularly injected with 10 ug of LNP formulated STARR™transcripts, STARR™ FLuc or TA STARR™, on day 0 and day 7. On day 16,the spleens were harvested and the splenocytes were isolated.Splenocytes (2.5×105 cells) were incubated with or without AH1A5(SPSYAYHQF) (SEQ ID NO: 111), beta-gal peptide (TPHPARIGL) (SEQ IDNO:112) at 1 ug/ml, and 1× Concanavalin A (Life Technologies). ELiSpotdetecting murine IFN-gamma (ImmunoSpot) was performed according to themanufacturer's instructions. As can be seen in FIG. 19 , TA STARR™elicited antigen-specific IFN-gamma responses.

BALB/c mice, 10 week-old female, were subcutaneously implanted in theright flank with 5×105 cells of CT26 cells in PBS. A day later,LNP-formulated STARR™ RNA was injected intramuscularly in the left legat a dose of 10 ug in 100 ul. The mice were administered another boostershot on day 8 with the same dose. For a group with combination treatmentof anti-mouse PD1 (RMP1-14, BioXCell) and anti-mouse PDL1 (10F.9G2,BioXcell), the combined checkpoint inhibitor (100 ug each) wasadministered via intraperitoneal injection in the right quadrant twiceweekly for two weeks starting on day 3. For a group with the treatmentof anti-mouse CTLA4 (9H10, BioxCell), 200 ug of the checkpoint inhibitorwas administered in the same manner but starting on day 7. Five mice ofthe group with the combo treatment of TA STARR™ vaccine and thecheckpoint inhibitors remained tumor-free on day 25, and were furtherchallenged by subcutaneous implantation of CT26 (5×105 cells) in theright flank where the implantation site was slightly above the firstimplantation site. Naïve mice were used as a control group. The tumorgrowth was monitored for another 17 days (i.e. up to day 42 since thefirst CT26 implantation) before euthanization. FIGS. 20A-20F illustratesreduced tumor growth resulting from TA STARR™ vaccination and FIG. 21shows prolonged protection resulting from treatment with the TA STARR™vaccine in combination with checkpoint inhibitors.

Splenocytes from the combination treatment group with TA STARR™ andanti-PD1/PDL1 were harvested for tetramer staining with AH1 peptide.Splenocytes from the control group with the LNP formulation buffer withthe same dosing schedule were used as a negative control. Thesplenocytes (2×106 cells) were incubated with AH1 (H-2Ld)-tetramer (MBL)followed by appropriate fluorescent-labeled antibodies (Alexa Fluor 488anti-CD8a (53-6.7), Pacific Orange anti-CD4 (RM4-5), and Pacific Blueanti-mouse CD3ε (145-2C11), eBioscience) and DRAQ7 (Invitrogen) byfollowing the manufacture's recommendation, and 500K events wereanalyzed by ZE5 Cell Analyzer (Bio-Rad). Results are shown in FIGS.22A-22C.

TABLE 9 Transgene ORF nucleotide sequence RNA mARM back # bone TransgeneSequence 2809 STARR^(TM) FlucAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACC (SEQCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAA IDGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAU NO:84)AUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGACAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGUCCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAA 2842 SINV FlucAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACC (SEQ repliconCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAA IDGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAU NO:85)AUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGACAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGUCCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAA 1782 mRNA Fluc AUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACC (SEQ  (TEV-CACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAA ID XbG)GCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAU NO:86)AUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGACAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGUCCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAA 2847 STARR^(TM) KRASAUGAAGUUGGUGGUUGUGGGGGCCGGGGGUGUUGGCAAAAGCGCCC (SEQ epitope wtUUACAAUUUGA ID NO:87) 2862 SINV EmptyAUGGAUCCUAGACGCUACGCCCCAAUGAUCCGACCAGCAAAACUCG (SEQ repliconAUGUACUUCCGAGGAACUGA ID NO:88) 3060 STARR^(TM) SignalAUGAGAGUGACAGCCCCUAGAACCUUACUGCUUCUGCUUUGGGGAG (SEQ peptide-CUGUUGCUCUGACAGAGACAUGGGCUGGAUCUCUGAGCGAGGUGAC ID gp70 withCGGCCAGGGCCUGUGCAUCGGCGCCGUGCCCAAGACCCACCAGGUG NO:89) AH1A5-CUGUGCAACACCACCCAGAAGACCAGCGACGGCAGCUACUACCUGG MITDCCGCUCCCACCGGCACCACCUGGGCCUGCAGCACCGGCCUGACCCCUUGCAUCAGCACCACCAUCCUGAACCUGACCACCGACUACUGCGUGCUGGUGGAGCUGUGGCCCAGGGUGACCUACCACAGCCCCAGCUACGCCUACCACCAGUUCGAGAGGAGGGCCAAGUACAAGAGGGAGCCCGUGAGCCUGACCCUGGCCCUGCUGCUGGGCGGCCUGACAAUGGGCGGCAUCGCCGCCGGCGUGGGCACCGGCACCACCGCCCUGGUGGCCACCCAGCAGUUCCAGCAGCUGCAGGCCGCCAUGCACGACGACCUGAAGGAGGUGGAGAAGUCCAUCACCAACCUGGAGAAGUCCCUGACCAGCCUGAGCGAGGUGGUGCUGCAGAACAGGAGGGGCCUGGACCUGCUGUUCCUGAAGGAGGGCGGCCUGUGCGCCGCCCUGAAGGAGGAGUGCUGCCUGUACGCCGACCACACCGGCCUGGUGAUCGUGGGCAUUGUCGCUGGCCUGGCCGUCCUCGCCGUGGUGGUGAUUGGAGCUGUGGUCGCAGCUGUUAUGUGCAGAAGAAAGUCAUCCGGCGGAAAGGGAGGCUCCUACUCUCAGGCUGCUUCUGCUACAGUGCCUAGAGCUCUUAUGUGUUUAUCU CAGCUGUAA 3061 STARR^(TM)Signal AUGAGAGUGACAGCCCCUAGAACCUUACUGCUUCUGCUUUGGGGAG (SEQ peptide-CUGUUGCUCUGACAGAGACAUGGGCUGGAUCUUACCACAGCCCCAG ID AH1A5 OVA-CUACGCCUACCACCAGUUCGAGAGGGGGGGAGGAGGCUCCGGGGGA NO:90) MITDGGAGGCUCCCUGAAGAUCAGCCAGGCCGUGCACGCCGCCCACGCCGAGAUCAACGAGGCCGGCCGGGAGGUGAUCGUGGGCAUUGUCGCUGGCCUGGCCGUCCUCGCCGUGGUGGUGAUUGGAGCUGUGGUCGCAGCUGUUAUGUGCAGAAGAAAGUCAUCCGGCGGAAAGGGAGGCUCCUACUCUCAGGCUGCUUCUGCUACAGUGCCUAGAGCUCUUAUGUGUUUAUC UCAGCUGUAA 3076STARR^(TM) Signal AUGAGAGUGACAGCCCCUAGAACCUUACUGCUUCUGCUUUGGGGAG (SEQpeptide- CUGUUGCUCUGACAGAGACAUGGGCUGGAUCUCUGAGCGAGGUGAC ID gp70 withCGGCCAGGGCCUGUGCAUCGGCGCCGUGCCCAAGACCCACCAGGUG NO:91) AH1A5-CUGUGCAACACCACCCAGAAGACCAGCGACGGCAGCUACUACCUGG MITD-FLAGCCGCUCCCACCGGCACCACCUGGGCCUGCAGCACCGGCCUGACCCCUUGCAUCAGCACCACCAUCCUGAACCUGACCACCGACUACUGCGUGCUGGUGGAGCUGUGGCCCAGGGUGACCUACCACAGCCCCAGCUACGCCUACCACCAGUUCGAGAGGAGGGCCAAGUACAAGAGGGAGCCCGUGAGCCUGACCCUGGCCCUGCUGCUGGGCGGCCUGACAAUGGGCGGCAUCGCCGCCGGCGUGGGCACCGGCACCACCGCCCUGGUGGCCACCCAGCAGUUCCAGCAGCUGCAGGCCGCCAUGCACGACGACCUGAAGGAGGUGGAGAAGUCCAUCACCAACCUGGAGAAGUCCCUGACCAGCCUGAGCGAGGUGGUGCUGCAGAACAGGAGGGGCCUGGACCUGCUGUUCCUGAAGGAGGGCGGCCUGUGCGCCGCCCUGAAGGAGGAGUGCUGCCUGUACGCCGACCACACCGGCCUGGUGAUCGUGGGCAUUGUCGCUGGCCUGGCCGUCCUCGCCGUGGUGGUGAUUGGAGCUGUGGUCGCAGCUGUUAUGUGCAGAAGAAAGUCAUCCGGCGGAAAGGGAGGCUCCUACUCUCAGGCUGCUUCUGCUACAGUGCCUAGAGCUCUUAUGUGUUUAUCUCAGCUGGGCGGCGGAGGCAGCGACUACAAGGACGACGAUGACAAGU AA 3068 STARR SignalAUGAGAGUGACAGCCCCUAGAACCUUACUGCUUCUGCUUUGGGGAG (SEQ peptide-CUGUUGCUCUGACAGAGACAUGGGCUGGAUCUUACCACAGCCCCAG ID AH1A5 OVA-CUACGCCUACCACCAGUUCGAGAGGGGGGGAGGAGGCUCCGGGGGA NO:92) MITD-FLAGGGAGGCUCCCUGAAGAUCAGCCAGGCCGUGCACGCCGCCCACGCCGAGAUCAACGAGGCCGGCCGGGAGGUGAUCGUGGGCAUUGUCGCUGGCCUGGCCGUCCUCGCCGUGGUGGUGAUUGGAGCUGUGGUCGCAGCUGUUAUGUGCAGAAGAAAGUCAUCCGGCGGAAAGGGAGGCUCCUACUCUCAGGCUGCUUCUGCUACAGUGCCUAGAGCUCUUAUGUGUUUAUCUCAGCUGGGCGGCGGAGGCAGCGACUACAAGGACGACGAUGACAAG UAATransgene ORF amino acid sequence mARM transgene # description Sequence2809, Fluc MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH 2842,IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPV 1782LGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILN (SEQVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPE IDSFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQ NO:93)IIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV* 2847 KRASMKLVVVGAGGVGKSALTI* (SEQ epitope wt ID NO:94) 2862 EmptyMDPRRYAPMIRPAKLDVLPRN* (SEQ ID NO:95) 3060 SignalMRVTAPRTLLLLLWGAVALTETWAGSLSEVTGQGLCIGAVPKTHQV (SEQ peptide-LCNTTQKTSDGSYYLAAPTGTTWACSTGLTPCISTTILNLTTDYCV ID gp70 withLVELWPRVTYHSPSYAYHQFERRAKYKREPVSLTLALLLGGLTMGG NO:96) AH1A5-IAAGVGTGTTALVATQQFQQLQAAMHDDLKEVEKSITNLEKSLTSL MITDSEVVLQNRRGLDLLFLKEGGLCAALKEECCLYADHTGLVIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSYSQAASATVPRALMCLS QL* 3061 SignalMRVTAPRTLLLLLWGAVALTETWAGSYHSPSYAYHQFERGGGGSGG (SEQ peptide-GGSLKISQAVHAAHAEINEAGREVIVGIVAGLAVLAVVVIGAVVAA ID AH1A5 OVA-VMCRRKSSGGKGGSYSQAASATVPRALMCLSQL* NO:97) MITD 3076 SignalMRVTAPRTLLLLLWGAVALTETWAGSLSEVTGQGLCIGAVPKTHQV (SEQ peptide-LCNTTQKTSDGSYYLAAPTGTTWACSTGLTPCISTTILNLTTDYCV ID gp70 withLVELWPRVTYHSPSYAYHQFERRAKYKREPVSLTLALLLGGLTMGG NO:98) AH1A5-IAAGVGTGTTALVATQQFQQLQAAMHDDLKEVEKSITNLEKSLTSL MITD-FLAGSEVVLQNRRGLDLLFLKEGGLCAALKEECCLYADHTGLVIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSYSQAASATVPRALMCLS QLGGGGSDYKDDDDK* 3068Signal MRVTAPRTLLLLLWGAVALTETWAGSYHSPSYAYHQFERGGGGSGG (SEQ peptide-GGSLKISQAVHAAHAEINEAGREVIVGIVAGLAVLAVVVIGAVVAA NO:99) AH1A5 OVA-VMCRRKSSGGKGGSYSQAASATVPRALMCLSQLGGGGSDYKDDDDK MITD-FLAG *whole RNA sequence mARM brief # name Sequence 2809 STARR^(TM) 2809AUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAAAU (SEQ FlucGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGA IDGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGG NO:100)UCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCCGUCGACGGCCCCACCAGCCUGUACCACCAGGCCAACAAGGGCGUGAGGGUGGCCUACUGGAUCGGCUUCGACACCACACCCUUCAUGUUCAAGAACCUGGCCGGCGCCUACCCCAGCUACAGCACCAACUGGGCCGACGAGACCGUGCUGACCGCCAGGAACAUCGGCCUGUGCAGCAGCGACGUGAUGGAGAGGAGCCGGAGAGGCAUGAGCAUCCUGAGGAAGAAAUACCUGAAGCCCAGCAACAACGUGCUGUUCAGCGUGGGCAGCACCAUCUACCACGAGAAGAGGGACCUGCUCAGGAGCUGGCACCUGCCCAGCGUGUUCCACCUGAGGGGCAAGCAGAACUACACCUGCAGGUGCGAGACCAUCGUGAGCUGCGACGGCUACGUGGUGAAGAGGAUCGCCAUCAGCCCCGGCCUGUACGGCAAGCCCAGCGGCUACGCCGCUACAAUGCACAGGGAGGGCUUCCUGUGCUGCAAGGUGACCGACACCCUGAACGGCGAGAGGGUGAGCUUCCCCGUGUGCACCUACGUGCCCGCCACCCUGUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGAGCGCCGACGACGCCCAGAAGCUGCUCGUGGGCCUGAACCAGAGGAUCGUGGUCAACGGCAGGACCCAGAGGAACACCAACACAAUGAAGAACUACCUGCUGCCCGUGGUGGCCCAGGCUUUCGCCAGGUGGGCCAAGGAGUACAAGGAGGACCAGGAAGACGAGAGGCCCCUGGGCCUGAGGGACAGGCAGCUGGUGAUGGGCUGCUGCUGGGCCUUCAGGCGGCACAAGAUCACCAGCAUCUACAAGAGGCCCGACACCCAGACCAUCAUCAAGGUGAACAGCGACUUCCACAGCUUCGUGCUGCCCAGGAUCGGCAGCAACACCCUGGAGAUCGGCCUGAGGACCCGGAUCAGGAAGAUGCUGGAGGAACACAAGGAGCCCAGCCCACUGAUCACCGCCGAGGACGUGCAGGAGGCCAAGUGCGCUGCCGACGAGGCCAAGGAGGUGAGGGAGGCCGAGGAACUGAGGGCCGCCCUGCCACCCCUGGCUGCCGACGUGGAGGAACCCACCCUGGAAGCCGACGUGGACCUGAUGCUGCAGGAGGCCGGCGCCGGAAGCGUGGAGACACCCAGGGGCCUGAUCAAGGUGACCAGCUACGACGGCGAGGACAAGAUCGGCAGCUACGCCGUGCUGAGCCCACAGGCCGUGCUGAAGUCCGAGAAGCUGAGCUGCAUCCACCCACUGGCCGAGCAGGUGAUCGUGAUCACCCACAGCGGCAGGAAGGGCAGGUACGCCGUGGAGCCCUACCACGGCAAGGUGGUCGUGCCCGAGGGCCACGCCAUCCCCGUGCAGGACUUCCAGGCCCUGAGCGAGAGCGCCACCAUCGUGUACAACGAGAGGGAGUUCGUGAACAGGUACCUGCACCAUAUCGCCACCCACGGCGGAGCCCUGAACACCGACGAGGAAUACUACAAGACCGUGAAGCCCAGCGAGCACGACGGCGAGUACCUGUACGACAUCGACAGGAAGCAGUGCGUGAAGAAAGAGCUGGUGACCGGCCUGGGACUGACCGGCGAGCUGGUGGACCCACCCUUCCACGAGUUCGCCUACGAGAGCCUGAGGACCAGACCCGCCGCUCCCUACCAGGUGCCCACCAUCGGCGUGUACGGCGUGCCCGGCAGCGGAAAGAGCGGCAUCAUCAAGAGCGCCGUGACCAAGAAAGACCUGGUGGUCAGCGCCAAGAAAGAGAACUGCGCCGAGAUCAUCAGGGACGUGAAGAAGAUGAAAGGCCUGGACGUGAACGCGCGCACCGUGGACAGCGUGCUGCUGAACGGCUGCAAGCACCCCGUGGAGACCCUGUACAUCGACGAGGCCUUCGCUUGCCACGCCGGCACCCUGAGGGCCCUGAUCGCCAUCAUCAGGCCCAAGAAAGCCGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUGAUGUGCCUGAAGGUGCACUUCAACCACGAGAUCUGCACCCAGGUGUUCCACAAGAGCAUCAGCAGGCGGUGCACCAAGAGCGUGACCAGCGUCGUGAGCACCCUGUUCUACGACAAGAAAAUGAGGACCACCAACCCCAAGGAGACCAAAAUCGUGAUCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCUGAUCCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCAGAUCGACUACAAGGGCAACGAGAUCAUGACCGCCGCUGCCAGCCAGGGCCUGACCAGGAAGGGCGUGUACGCCGUGAGGUACAAGGUGAACGAGAACCCACUGUACGCUCCCACCAGCGAGCACGUGAACGUGCUGCUGACCAGGACCGAGGACAGGAUCGUGUGGAAGACCCUGGCCGGCGACCCCUGGAUCAAGACCCUGACCGCCAAGUACCCCGGCAACUUCACCGCCACCAUCGAAGAGUGGCAGGCCGAGCACGACGCCAUCAUGAGGCACAUCCUGGAGAGGCCCGACCCCACCGACGUGUUCCAGAACAAGGCCAACGUGUGCUGGGCCAAGGCCCUGGUGCCCGUGCUGAAGACCGCCGGCAUCGACAUGACCACAGAGCAGUGGAACACCGUGGACUACUUCGAGACCGACAAGGCCCACAGCGCCGAGAUCGUGCUGAACCAGCUGUGCGUGAGGUUCUUCGGCCUGGACCUGGACAGCGGCCUGUUCAGCGCCCCCACCGUGCCACUGAGCAUCAGGAACAACCACUGGGACAACAGCCCCAGCCCAAACAUGUACGGCCUGAACAAGGAGGUGGUCAGGCAGCUGAGCAGGCGGUACCCACAGCUGCCCAGGGCCGUGGCCACCGGCAGGGUGUACGACAUGAACACCGGCACCCUGAGGAACUACGACCCCAGGAUCAACCUGGUGCCCGUGAACAGGCGGCUGCCCCACGCCCUGGUGCUGCACCACAACGAGCACCCACAGAGCGACUUCAGCUCCUUCGUGAGCAAGCUGAAAGGCAGGACCGUGCUGGUCGUGGGCGAGAAGCUGAGCGUGCCCGGCAAGAUGGUGGACUGGCUGAGCGACAGGCCCGAGGCCACCUUCCGGGCCAGGCUGGACCUCGGCAUCCCCGGCGACGUGCCCAAGUACGACAUCAUCUUCGUGAACGUCAGGACCCCAUACAAGUACCACCAUUACCAGCAGUGCGAGGACCACGCCAUCAAGCUGAGCAUGCUGACCAAGAAGGCCUGCCUGCACCUGAACCCCGGAGGCACCUGCGUGAGCAUCGGCUACGGCUACGCCGACAGGGCCAGCGAGAGCAUCAUUGGCGCCAUCGCCAGGCUGUUCAAGUUCAGCAGGGUGUGCAAACCCAAGAGCAGCCUGGAGGAAACCGAGGUGCUGUUCGUGUUCAUCGGCUACGACCGGAAGGCCAGGACCCACAACCCCUACAAGCUGAGCAGCACCCUGACAAACAUCUACACCGGCAGCAGGCUGCACGAGGCCGGCUGCGCCCCCAGCUACCACGUGGUCAGGGGCGAUAUCGCCACCGCCACCGAGGGCGUGAUCAUCAACGCUGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGUGUGCGGCGCCCUGUACAAGAAGUUCCCCGAGAGCUUCGACCUGCAGCCCAUCGAGGUGGGCAAGGCCAGGCUGGUGAAGGGCGCCGCUAAGCACAUCAUCCACGCCGUGGGCCCCAACUUCAACAAGGUGAGCGAGGUGGAAGGCGACAAGCAGCUGGCCGAAGCCUACGAGAGCAUCGCCAAGAUCGUGAACGACAAUAACUACAAGAGCGUGGCCAUCCCACUGCUCAGCACCGGCAUCUUCAGCGGCAACAAGGACAGGCUGACCCAGAGCCUGAACCACCUGCUCACCGCCCUGGACACCACCGAUGCCGACGUGGCCAUCUACUGCAGGGACAAGAAGUGGGAGAUGACCCUGAAGGAGGCCGUGGCCAGGCGGGAGGCCGUGGAAGAGAUCUGCAUCAGCGACGACUCCAGCGUGACCGAGCCCGACGCCGAGCUGGUGAGGGUGCACCCCAAGAGCUCCCUGGCCGGCAGGAAGGGCUACAGCACCAGCGACGGCAAGACCUUCAGCUACCUGGAGGGCACCAAGUUCCACCAGGCCGCUAAGGACAUCGCCGAGAUCAACGCUAUGUGGCCCGUGGCCACCGAGGCCAACGAGCAGGUGUGCAUGUACAUCCUGGGCGAGAGCAUGUCCAGCAUCAGGAGCAAGUGCCCCGUGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCUGCCCUGCCUGUGCAUCCACGCUAUGACACCCGAGAGGGUGCAGCGGCUGAAGGCCAGCAGGCCCGAGCAGAUCACCGUGUGCAGCUCCUUCCCACUGCCCAAGUACAGGAUCACCGGCGUGCAGAAGAUCCAGUGCAGCCAGCCCAUCCUGUUCAGCCCAAAGGUGCCCGCCUACAUCCACCCCAGGAAGUACCUGGUGGAGACCCCACCCGUGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCUGAUCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCAUCAUUAUCGAGGAAGAGGAAGAGGACAGCAUCAGCCUGCUGAGCGACGGCCCCACCCACCAGGUGCUGCAGGUGGAGGCCGACAUCCACGGCCCACCCAGCGUGUCCAGCUCCAGCUGGAGCAUCCCACACGCCAGCGACUUCGACGUGGACAGCCUGAGCAUCCUGGACACCCUGGAGGGCGCCAGCGUGACCUCCGGCGCCACCAGCGCCGAGACCAACAGCUACUUCGCCAAGAGCAUGGAGUUCCUGGCCAGGCCCGUGCCAGCUCCCAGGACCGUGUUCAGGAACCCACCCCACCCAGCUCCCAGGACCAGGACCCCAAGCCUGGCUCCCAGCAGGGCCUGCAGCAGGACCAGCCUGGUGAGCACCCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAACUGGAGGCCCUGACACCCAGCAGGACCCCCAGCAGGUCCGUGAGCAGGACUAGUCUGGUGUCCAACCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAAUUCGAGGCCUUCGUGGCCCAGCAACAGAGACGGUUCGACGCCGGCGCCUACAUCUUCAGCAGCGACACCGGCCAGGGACACCUGCAGCAAAAGAGCGUGAGGCAGACCGUGCUGAGCGAGGUGGUGCUGGAGAGGACCGAGCUGGAAAUCAGCUACGCCCCCAGGCUGGACCAGGAGAAGGAGGAACUGCUCAGGAAGAAACUGCAGCUGAACCCCACCCCAGCCAACAGGAGCAGGUACCAGAGCAGGAAGGUGGAGAACAUGAAGGCCAUCACCGCCAGGCGGAUCCUGCAGGGCCUGGGACACUACCUGAAGGCCGAGGGCAAGGUGGAGUGCUACAGGACCCUGCACCCCGUGCCACUGUACAGCUCCAGCGUGAACAGGGCCUUCUCCAGCCCCAAGGUGGCCGUGGAGGCCUGCAACGCUAUGCUGAAGGAGAACUUCCCCACCGUGGCCAGCUACUGCAUCAUCCCCGAGUACGACGCCUACCUGGACAUGGUGGACGGCGCCAGCUGCUGCCUGGACACCGCCAGCUUCUGCCCCGCCAAGCUGAGGAGCUUCCCCAAGAAACACAGCUACCUGGAGCCCACCAUCAGGAGCGCCGUGCCCAGCGCCAUCCAGAACACCCUGCAGAACGUGCUGGCCGCUGCCACCAAGAGGAACUGCAACGUGACCCAGAUGAGGGAGCUGCCCGUGCUGGACAGCGCUGCCUUCAACGUGGAGUGCUUCAAGAAAUACGCCUGCAACAACGAGUACUGGGAGACCUUCAAGGAGAACCCCAUCAGGCUGACCGAAGAGAACGUGGUGAACUACAUCACCAAGCUGAAGGGCCCCAAGGCCGCUGCCCUGUUCGCUAAGACCCACAACCUGAACAUGCUGCAGGACAUCCCAAUGGACAGGUUCGUGAUGGACCUGAAGAGGGACGUGAAGGUGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGUGCAGGUGAUCCAGGCCGCUGACCCACUGGCCACCGCCUACCUGUGCGGCAUCCACAGGGAGCUGGUGAGGCGGCUGAACGCCGUGCUGCUGCCCAACAUCCACACCCUGUUCGACAUGAGCGCCGAGGACUUCGACGCCAUCAUCGCCGAGCACUUCCAGCCCGGCGACUGCGUGCUGGAGACCGACAUCGCCAGCUUCGACAAGAGCGAGGAUGACGCUAUGGCCCUGACCGCUCUGAUGAUCCUGGAGGACCUGGGCGUGGACGCCGAGCUGCUCACCCUGAUCGAGGCUGCCUUCGGCGAGAUCAGCUCCAUCCACCUGCCCACCAAGACCAAGUUCAAGUUCGGCGCUAUGAUGAAAAGCGGAAUGUUCCUGACCCUGUUCGUGAACACCGUGAUCAACAUUGUGAUCGCCAGCAGGGUGCUGCGGGAGAGGCUGACCGGCAGCCCCUGCGCUGCCUUCAUCGGCGACGACAACAUCGUGAAGGGCGUGAAAAGCGACAAGCUGAUGGCCGACAGGUGCGCCACCUGGCUGAACAUGGAGGUGAAGAUCAUCGACGCCGUGGUGGGCGAGAAGGCCCCCUACUUCUGCGGCGGAUUCAUCCUGUGCGACAGCGUGACCGGCACCGCCUGCAGGGUGGCCGACCCCCUGAAGAGGCUGUUCAAGCUGGGCAAGCCACUGGCCGCUGACGAUGAGCACGACGAUGACAGGCGGAGGGCCCUGCACGAGGAAAGCACCAGGUGGAACAGGGUGGGCAUCCUGAGCGAGCUGUGCAAGGCCGUGGAGAGCAGGUACGAGACCGUGGGCACCAGCAUCAUCGUGAUGGCUAUGACCACACUGGCCAGCUCCGUCAAGAGCUUCUCCUACCUGAGGGGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGGCCGCCACCAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGACAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGUCCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAACUCGAGUAUGUUACGUGCAAAGGUGAUUGUCACCCCCCGAAAGACCAUAUUGUGACACACCCUCAGUAUCACGCCCAAACAUUUACAGCCGCGGUGUCAAAAACCGCGUGGACGUGGUUAACAUCCCUGCUGGGAGGAUCAGCCGUAAUUAUUAUAAUUGGCUUGGUGCUGGCUACUAUUGUGGCCAUGUACGUGCUGACCAACCAGAAACAUAAUUGAAUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 2842 SINV 2842AUUGACGGCGUAGUACACACUAUUGAAUCAAACAGCCGACCAAUUG (SEQ FlucCACUACCAUCACAAUGGAGAAGCCAGUAGUAAACGUAGACGUAGAC IDCCCCAGAGUCCGUUUGUCGUGCAACUGCAAAAAAGCUUCCCGCAAU NO:101)UUGAGGUAGUAGCACAGCAGGUCACUCCAAAUGACCAUGCUAAUGCCAGAGCAUUUUCGCAUCUGGCCAGUAAACUAAUCGAGCUGGAGGUUCCUACCACAGCGACGAUCUUGGACAUAGGCAGCGCACCGGCUCGUAGAAUGUUUUCCGAGCACCAGUAUCAUUGUGUCUGCCCCAUGCGUAGUCCAGAAGACCCGGACCGCAUGAUGAAAUAUGCCAGUAAACUGGCGGAAAAAGCGUGCAAGAUUACAAACAAGAACUUGCAUGAGAAGAUUAAGGAUCUCCGGACCGUACUUGAUACGCCGGAUGCUGAAACACCAUCGCUCUGCUUUCACAACGAUGUUACCUGCAACAUGCGUGCCGAAUAUUCCGUCAUGCAGGACGUGUAUAUCAACGCUCCCGGAACUAUCUAUCAUCAGGCUAUGAAAGGCGUGCGGACCCUGUACUGGAUUGGCUUCGACACCACCCAGUUCAUGUUCUCGGCUAUGGCAGGUUCGUACCCUGCGUACAACACCAACUGGGCCGACGAGAAAGUCCUUGAAGCGCGUAACAUCGGACUUUGCAGCACAAAGCUGAGUGAAGGUAGGACAGGAAAAUUGUCGAUAAUGAGGAAGAAGGAGUUGAAGCCCGGGUCGCGGGUUUAUUUCUCCGUAGGAUCGACACUUUAUCCAGAACACAGAGCCAGCUUGCAGAGCUGGCAUCUUCCAUCGGUGUUCCACUUGAAUGGAAAGCAGUCGUACACUUGCCGCUGUGAUACAGUGGUGAGUUGCGAAGGCUACGUAGUGAAGAAAAUCACCAUCAGUCCCGGGAUCACGGGAGAAACCGUGGGAUACGCGGUUACACACAAUAGCGAGGGCUUCUUGCUAUGCAAAGUUACUGACACAGUAAAAGGAGAACGGGUAUCGUUCCCUGUGUGCACGUACAUCCCGGCCACCAUAUGCGAUCAGAUGACUGGUAUAAUGGCCACGGAUAUAUCACCUGACGAUGCACAAAAACUUCUGGUUGGGCUCAACCAGCGAAUUGUCAUUAACGGUAGGACUAACAGGAACACCAACACCAUGCAAAAUUACCUUCUGCCGAUCAUAGCACAAGGGUUCAGCAAAUGGGCUAAGGAGCGCAAGGAUGAUCUUGAUAACGAGAAAAUGCUGGGUACUAGAGAACGCAAGCUUACGUAUGGCUGCUUGUGGGCGUUUCGCACUAAGAAAGUACAUUCGUUUUAUCGCCCACCUGGAACGCAGACCUGCGUAAAAGUCCCAGCCUCUUUUAGCGCUUUUCCCAUGUCGUCCGUAUGGACGACCUCUUUGCCCAUGUCGCUGAGGCAGAAAUUGAAACUGGCAUUGCAACCAAAGAAGGAGGAAAAACUGCUGCAGGUCUCGGAGGAAUUAGUCAUGGAGGCCAAGGCUGCUUUUGAGGAUGCUCAGGAGGAAGCCAGAGCGGAGAAGCUCCGAGAAGCACUUCCACCAUUAGUGGCAGACAAAGGCAUCGAGGCAGCCGCAGAAGUUGUCUGCGAAGUGGAGGGGCUCCAGGCGGACAUCGGAGCAGCAUUAGUUGAAACCCCGCGCGGUCACGUAAGGAUAAUACCUCAAGCAAAUGACCGUAUGAUCGGACAGUAUAUCGUUGUCUCGCCAAACUCUGUGCUGAAGAAUGCCAAACUCGCACCAGCGCACCCGCUAGCAGAUCAGGUUAAGAUCAUAACACACUCCGGAAGAUCAGGAAGGUACGCGGUCGAACCAUACGACGCUAAAGUACUGAUGCCAGCAGGAGGUGCCGUACCAUGGCCAGAAUUCCUAGCACUGAGUGAGAGCGCCACGUUAGUGUACAACGAAAGAGAGUUUGUGAACCGCAAACUAUACCACAUUGCCAUGCAUGGCCCCGCCAAGAAUACAGAAGAGGAGCAGUACAAGGUUACAAAGGCAGAGCUUGCAGAAACAGAGUACGUGUUUGACGUGGACAAGAAGCGUUGCGUUAAGAAGGAAGAAGCCUCAGGUCUGGUCCUCUCGGGAGAACUGACCAACCCUCCCUAUCAUGAGCUAGCUCUGGAGGGACUGAAGACCCGACCUGCGGUCCCGUACAAGGUCGAAACAAUAGGAGUGAUAGGCACACCGGGGUCGGGCAAGUCAGCUAUUAUCAAGUCAACUGUCACGGCACGAGAUCUUGUUACCAGCGGAAAGAAAGAAAAUUGUCGCGAAAUUGAGGCCGACGUGCUAAGACUGAGGGGUAUGCAGAUUACGUCGAAGACAGUAGAUUCGGUUAUGCUCAACGGAUGCCACAAAGCCGUAGAAGUGCUGUACGUUGACGAAGCGUUCGCGUGCCACGCAGGAGCACUACUUGCCUUGAUUGCUAUCGUCAGGCCCCGCAAGAAGGUAGUACUAUGCGGAGACCCCAUGCAAUGCGGAUUCUUCAACAUGAUGCAACUAAAGGUACAUUUCAAUCACCCUGAAAAAGACAUAUGCACCAAGACAUUCUACAAGUAUAUCUCCCGGCGUUGCACACAGCCAGUUACAGCUAUUGUAUCGACACUGCAUUACGAUGGAAAGAUGAAAACCACGAACCCGUGCAAGAAGAACAUUGAAAUCGAUAUUACAGGGGCCACAAAGCCGAAGCCAGGGGAUAUCAUCCUGACAUGUUUCCGCGGGUGGGUUAAGCAAUUGCAAAUCGACUAUCCCGGACAUGAAGUAAUGACAGCCGCGGCCUCACAAGGGCUAACCAGAAAAGGAGUGUAUGCCGUCCGGCAAAAAGUCAAUGAAAACCCACUGUACGCGAUCACAUCAGAGCAUGUGAACGUGUUGCUCACCCGCACUGAGGACAGGCUAGUGUGGAAAACCUUGCAGGGCGACCCAUGGAUUAAGCAGCUCACUAACAUACCUAAAGGAAACUUUCAGGCUACUAUAGAGGACUGGGAAGCUGAACACAAGGGAAUAAUUGCUGCAAUAAACAGCCCCACUCCCCGUGCCAAUCCGUUCAGCUGCAAGACCAACGUUUGCUGGGCGAAAGCAUUGGAACCGAUACUAGCCACGGCCGGUAUCGUACUUACCGGUUGCCAGUGGAGCGAACUGUUCCCACAGUUUGCGGAUGACAAACCACAUUCGGCCAUUUACGCCUUAGACGUAAUUUGCAUUAAGUUUUUCGGCAUGGACUUGACAAGCGGACUGUUUUCUAAACAGAGCAUCCCACUAACGUACCAUCCCGCCGAUUCAGCGAGGCCGGUAGCUCAUUGGGACAACAGCCCAGGAACCCGCAAGUAUGGGUACGAUCACGCCAUUGCCGCCGAACUCUCCCGUAGAUUUCCGGUGUUCCAGCUAGCUGGGAAGGGCACACAACUUGAUUUGCAGACGGGGAGAACCAGAGUUAUCUCUGCACAGCAUAACCUGGUCCCGGUGAACCGCAAUCUUCCUCACGCCUUAGUCCCCGAGUACAAGGAGAAGCAACCCGGCCCGGUCGAAAAAUUCUUGAACCAGUUCAAACACCACUCAGUACUUGUGGUAUCAGAGGAAAAAAUUGAAGCUCCCCGUAAGAGAAUCGAAUGGAUCGCCCCGAUUGGCAUAGCCGGUGCAGAUAAGAACUACAACCUGGCUUUCGGGUUUCCGCCGCAGGCACGGUACGACCUGGUGUUCAUCAACAUUGGAACUAAAUACAGAAACCACCACUUUCAGCAGUGCGAAGACCAUGCGGCGACCUUAAAAACCCUUUCGCGUUCGGCCCUGAAUUGCCUUAACCCAGGAGGCACCCUCGUGGUGAAGUCCUAUGGCUACGCCGACCGCAACAGUGAGGACGUAGUCACCGCUCUUGCCAGAAAGUUUGUCAGGGUGUCUGCAGCGAGACCAGAUUGUGUCUCAAGCAAUACAGAAAUGUACCUGAUUUUCCGACAACUAGACAACAGCCGUACACGGCAAUUCACCCCGCACCAUCUGAAUUGCGUGAUUUCGUCCGUGUAUGAGGGUACAAGAGAUGGAGUUGGAGCCGCGCCGUCAUACCGCACCAAAAGGGAGAAUAUUGCUGACUGUCAAGAGGAAGCAGUUGUCAACGCAGCCAAUCCGCUGGGUAGACCAGGCGAAGGAGUCUGCCGUGCCAUCUAUAAACGUUGGCCGACCAGUUUUACCGAUUCAGCCACGGAGACAGGCACCGCAAGAAUGACUGUGUGCCUAGGAAAGAAAGUGAUCCACGCGGUCGGCCCUGAUUUCCGGAAGCACCCAGAAGCAGAAGCCUUGAAAUUGCUACAAAACGCCUACCAUGCAGUGGCAGACUUAGUAAAUGAACAUAACAUCAAGUCUGUCGCCAUUCCACUGCUAUCUACAGGCAUUUACGCAGCCGGAAAAGACCGCCUUGAAGUAUCACUUAACUGCUUGACAACCGCGCUAGACAGAACUGACGCGGACGUAACCAUCUAUUGCCUGGAUAAGAAGUGGAAGGAAAGAAUCGACGCGGCACUCCAACUUAAGGAGUCUGUAACAGAGCUGAAGGAUGAAGAUAUGGAGAUCGACGAUGAGUUAGUAUGGAUCCAUCCAGACAGUUGCUUGAAGGGAAGAAAGGGAUUCAGUACUACAAAAGGAAAAUUGUAUUCGUACUUCGAAGGCACCAAAUUCCAUCAAGCAGCAAAAGACAUGGCGGAGAUAAAGGUCCUGUUCCCUAAUGACCAGGAAAGUAAUGAACAACUGUGUGCCUACAUAUUGGGUGAGACCAUGGAAGCAAUCCGCGAAAAGUGCCCGGUCGACCAUAACCCGUCGUCUAGCCCGCCCAAAACGUUGCCGUGCCUUUGCAUGUAUGCCAUGACGCCAGAAAGGGUCCACAGACUUAGAAGCAAUAACGUCAAAGAAGUUACAGUAUGCUCCUCCACCCCCCUUCCUAAGCACAAAAUUAAGAAUGUUCAGAAGGUUCAGUGCACGAAAGUAGUCCUGUUUAAUCCGCACACUCCCGCAUUCGUUCCCGCCCGUAAGUACAUAGAAGUGCCAGAACAGCCUACCGCUCCUCCUGCACAGGCCGAGGAGGCCCCCGAAGUUGUAGCGACACCGUCACCAUCUACAGCUGAUAACACCUCGCUUGAUGUCACAGACAUCUCACUGGAUAUGGAUGACAGUAGCGAAGGCUCACUUUUUUCGAGCUUUAGCGGAUCGGACAACUCUAUUACUAGUAUGGACAGUUGGUCGUCAGGACCUAGUUCACUAGAGAUAGUAGACCGAAGGCAGGUGGUGGUGGCUGACGUUCAUGCCGUCCAAGAGCCUGCCCCUAUUCCACCGCCAAGGCUAAAGAAGAUGGCCCGCCUGGCAGCGGCAAGAAAAGAGCCCACUCCACCGGCAAGCAAUAGCUCUGAGUCCCUCCACCUCUCUUUUGGUGGGGUAUCCAUGUCCCUCGGAUCAAUUUUCGACGGAGAGACGGCCCGCCAGGCAGCGGUACAACCCCUGGCAACAGGCCCCACGGAUGUGCCUAUGUCUUUCGGAUCGUUUUCCGACGGAGAGAUUGAUGAGCUGAGCCGCAGAGUAACUGAGUCCGAACCCGUCCUGUUUGGAUCAUUUGAACCGGGCGAAGUGAACUCAAUUAUAUCGUCCCGAUCAGCCGUAUCUUUUCCUCUACGCAAGCAGAGACGUAGACGCAGGAGCAGGAGGACUGAAUACUGACUAACCGGGGUAGGUGGGUACAUAUUUUCGACGGACACAGGCCCUGGGCACUUGCAAAAGAAGUCCGUUCUGCAGAACCAGCUUACAGAACCGACCUUGGAGCGCAAUGUCCUGGAAAGAAUUCAUGCCCCGGUGCUCGACACGUCGAAAGAGGAACAACUCAAACUCAGGUACCAGAUGAUGCCCACCGAAGCCAACAAAAGUAGGUACCAGUCUCGUAAAGUAGAAAAUCAGAAAGCCAUAACCACUGAGCGACUACUGUCAGGACUACGACUGUAUAACUCUGCCACAGAUCAGCCAGAAUGCUAUAAGAUCACCUAUCCGAAACCAUUGUACUCCAGUAGCGUACCGGCGAACUACUCCGAUCCACAGUUCGCUGUAGCUGUCUGUAACAACUAUCUGCAUGAGAACUAUCCGACAGUAGCAUCUUAUCAGAUUACUGACGAGUACGAUGCUUACUUGGAUAUGGUAGACGGGACAGUCGCCUGCCUGGACACUGCAACCUUCUGCCCCGCUAAGCUUAGAAGUUACCCGAAAAAACAUGAGUAUAGAGCCCCGAAUAUCCGCAGUGCGGUUCCAUCAGCGAUGCAGAACACGCUACAAAAUGUGCUCAUUGCCGCAACUAAAAGAAAUUGCAACGUCACGCAGAUGCGUGAACUGCCAACACUGGACUCAGCGACAUUCAAUGUCGAAUGCUUUCGAAAAUAUGCAUGUAAUGACGAGUAUUGGGAGGAGUUCGCUCGGAAGCCAAUUAGGAUUACCACUGAGUUUGUCACCGCAUAUGUAGCUAGACUGAAAGGCCCUAAGGCCGCCGCACUAUUUGCAAAGACGUAUAAUUUGGUCCCAUUGCAAGAAGUGCCUAUGGAUAGAUUCGUCAUGGACAUGAAAAGAGACGUGAAAGUUACACCAGGCACGAAACACACAGAAGAAAGACCGAAAGUACAAGUGAUACAAGCCGCAGAACCCCUGGCGACUGCUUACUUAUGCGGGAUUCACCGGGAAUUAGUGCGUAGGCUUACGGCCGUCUUGCUUCCAAACAUUCACACGCUUUUUGACAUGUCGGCGGAGGAUUUUGAUGCAAUCAUAGCAGAACACUUCAAGCAAGGCGACCCGGUACUGGAGACGGAUAUCGCAUCAUUCGACAAAAGCCAAGACGACGCUAUGGCGUUAACCGGUCUGAUGAUCUUGGAGGACCUGGGUGUGGAUCAACCACUACUCGACUUGAUCGAGUGCGCCUUUGGAGAAAUAUCAUCCACCCAUCUACCUACGGGUACUCGUUUUAAAUUCGGGGCGAUGAUGAAAUCCGGAAUGUUCCUCACACUUUUUGUCAACACAGUUUUGAAUGUCGUUAUCGCCAGCAGAGUACUAGAGGAGCGGCUUAAAACGUCCAGAUGUGCAGCGUUCAUUGGCGACGACAACAUCAUACAUGGAGUAGUAUCUGACAAAGAAAUGGCUGAGAGGUGCGCCACCUGGCUCAACAUGGAGGUUAAGAUCAUCGACGCAGUCAUCGGUGAGAGACCACCUUACUUCUGCGGCGGAUUUAUCUUGCAAGAUUCGGUUACUUCCACAGCGUGCCGCGUGGCGGAUCCCCUGAAAAGGCUGUUUAAGUUGGGUAAACCGCUCCCAGCCGACGACGAGCAAGACGAAGACAGAAGACGCGCUCUGCUAGAUGAAACAAAGGCGUGGUUUAGAGUAGGUAUAACAGGCACUUUAGCAGUGGCCGUGACGACCCGGUAUGAGGUAGACAAUAUUACACCUGUCCUACUGGCAUUGAGAACUUUUGCCCAGAGCAAAAGAGCAUUCCAAGCCAUCAGAGGGGAAAUAAAGCAUCUCUACGGUGGUCCUAAAUAGUCAGCAUAGUACAUUUCAUCUGACUAAUACUACAACACCACCACCAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGACAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGUCCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAAACGCGUGCUAGACCAUGGAUCCUAGACGCUACGCCCCAAUGAUCCGACCAGCAAAACUCGAUGUACUUCCGAGGAACUGAUGUGCAUAAUGCAUCAGGCUGGUACAUUAGAUCCCCGCUUACCGCGGGCAAUAUAGCAACACUAAAAACUCGAUGUACUUCCGAGGAAGCGCAGUGCAUAAUGCUGCGCAGUGUUGCCACAUAACCACUAUAUUAACCAUUUAUCUAGCGGACGCCAAAAACUCAAUGUAUUUCUGAGGAAGCGUGGUGCAUAAUGCCACGCAGCGUCUGCAUAACUUUUAUUAUUUCUUUUAUUAAUCAACAAAAUUUUGUUUUUAACAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAA1782 mRNA 1782 AGGAAACUUAAGUCAACACAACAUAUACAAAACAAACGAAUCUCAA (SEQ FlucGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUU IDUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUUUACGAACGA NO:102)UAGCCAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGACAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGUCCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAACUCGAGCUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAUUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAA 2847STARR^(TM) 2847 AUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAAAU (SEQ KRASGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGA ID wtGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGG NO:103)UCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCCGUCGACGGCCCCACCAGCCUGUACCACCAGGCCAACAAGGGCGUGAGGGUGGCCUACUGGAUCGGCUUCGACACCACACCCUUCAUGUUCAAGAACCUGGCCGGCGCCUACCCCAGCUACAGCACCAACUGGGCCGACGAGACCGUGCUGACCGCCAGGAACAUCGGCCUGUGCAGCAGCGACGUGAUGGAGAGGAGCCGGAGAGGCAUGAGCAUCCUGAGGAAGAAAUACCUGAAGCCCAGCAACAACGUGCUGUUCAGCGUGGGCAGCACCAUCUACCACGAGAAGAGGGACCUGCUCAGGAGCUGGCACCUGCCCAGCGUGUUCCACCUGAGGGGCAAGCAGAACUACACCUGCAGGUGCGAGACCAUCGUGAGCUGCGACGGCUACGUGGUGAAGAGGAUCGCCAUCAGCCCCGGCCUGUACGGCAAGCCCAGCGGCUACGCCGCUACAAUGCACAGGGAGGGCUUCCUGUGCUGCAAGGUGACCGACACCCUGAACGGCGAGAGGGUGAGCUUCCCCGUGUGCACCUACGUGCCCGCCACCCUGUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGAGCGCCGACGACGCCCAGAAGCUGCUCGUGGGCCUGAACCAGAGGAUCGUGGUCAACGGCAGGACCCAGAGGAACACCAACACAAUGAAGAACUACCUGCUGCCCGUGGUGGCCCAGGCUUUCGCCAGGUGGGCCAAGGAGUACAAGGAGGACCAGGAAGACGAGAGGCCCCUGGGCCUGAGGGACAGGCAGCUGGUGAUGGGCUGCUGCUGGGCCUUCAGGCGGCACAAGAUCACCAGCAUCUACAAGAGGCCCGACACCCAGACCAUCAUCAAGGUGAACAGCGACUUCCACAGCUUCGUGCUGCCCAGGAUCGGCAGCAACACCCUGGAGAUCGGCCUGAGGACCCGGAUCAGGAAGAUGCUGGAGGAACACAAGGAGCCCAGCCCACUGAUCACCGCCGAGGACGUGCAGGAGGCCAAGUGCGCUGCCGACGAGGCCAAGGAGGUGAGGGAGGCCGAGGAACUGAGGGCCGCCCUGCCACCCCUGGCUGCCGACGUGGAGGAACCCACCCUGGAAGCCGACGUGGACCUGAUGCUGCAGGAGGCCGGCGCCGGAAGCGUGGAGACACCCAGGGGCCUGAUCAAGGUGACCAGCUACGACGGCGAGGACAAGAUCGGCAGCUACGCCGUGCUGAGCCCACAGGCCGUGCUGAAGUCCGAGAAGCUGAGCUGCAUCCACCCACUGGCCGAGCAGGUGAUCGUGAUCACCCACAGCGGCAGGAAGGGCAGGUACGCCGUGGAGCCCUACCACGGCAAGGUGGUCGUGCCCGAGGGCCACGCCAUCCCCGUGCAGGACUUCCAGGCCCUGAGCGAGAGCGCCACCAUCGUGUACAACGAGAGGGAGUUCGUGAACAGGUACCUGCACCAUAUCGCCACCCACGGCGGAGCCCUGAACACCGACGAGGAAUACUACAAGACCGUGAAGCCCAGCGAGCACGACGGCGAGUACCUGUACGACAUCGACAGGAAGCAGUGCGUGAAGAAAGAGCUGGUGACCGGCCUGGGACUGACCGGCGAGCUGGUGGACCCACCCUUCCACGAGUUCGCCUACGAGAGCCUGAGGACCAGACCCGCCGCUCCCUACCAGGUGCCCACCAUCGGCGUGUACGGCGUGCCCGGCAGCGGAAAGAGCGGCAUCAUCAAGAGCGCCGUGACCAAGAAAGACCUGGUGGUCAGCGCCAAGAAAGAGAACUGCGCCGAGAUCAUCAGGGACGUGAAGAAGAUGAAAGGCCUGGACGUGAACGCGCGCACCGUGGACAGCGUGCUGCUGAACGGCUGCAAGCACCCCGUGGAGACCCUGUACAUCGACGAGGCCUUCGCUUGCCACGCCGGCACCCUGAGGGCCCUGAUCGCCAUCAUCAGGCCCAAGAAAGCCGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUGAUGUGCCUGAAGGUGCACUUCAACCACGAGAUCUGCACCCAGGUGUUCCACAAGAGCAUCAGCAGGCGGUGCACCAAGAGCGUGACCAGCGUCGUGAGCACCCUGUUCUACGACAAGAAAAUGAGGACCACCAACCCCAAGGAGACCAAAAUCGUGAUCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCUGAUCCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCAGAUCGACUACAAGGGCAACGAGAUCAUGACCGCCGCUGCCAGCCAGGGCCUGACCAGGAAGGGCGUGUACGCCGUGAGGUACAAGGUGAACGAGAACCCACUGUACGCUCCCACCAGCGAGCACGUGAACGUGCUGCUGACCAGGACCGAGGACAGGAUCGUGUGGAAGACCCUGGCCGGCGACCCCUGGAUCAAGACCCUGACCGCCAAGUACCCCGGCAACUUCACCGCCACCAUCGAAGAGUGGCAGGCCGAGCACGACGCCAUCAUGAGGCACAUCCUGGAGAGGCCCGACCCCACCGACGUGUUCCAGAACAAGGCCAACGUGUGCUGGGCCAAGGCCCUGGUGCCCGUGCUGAAGACCGCCGGCAUCGACAUGACCACAGAGCAGUGGAACACCGUGGACUACUUCGAGACCGACAAGGCCCACAGCGCCGAGAUCGUGCUGAACCAGCUGUGCGUGAGGUUCUUCGGCCUGGACCUGGACAGCGGCCUGUUCAGCGCCCCCACCGUGCCACUGAGCAUCAGGAACAACCACUGGGACAACAGCCCCAGCCCAAACAUGUACGGCCUGAACAAGGAGGUGGUCAGGCAGCUGAGCAGGCGGUACCCACAGCUGCCCAGGGCCGUGGCCACCGGCAGGGUGUACGACAUGAACACCGGCACCCUGAGGAACUACGACCCCAGGAUCAACCUGGUGCCCGUGAACAGGCGGCUGCCCCACGCCCUGGUGCUGCACCACAACGAGCACCCACAGAGCGACUUCAGCUCCUUCGUGAGCAAGCUGAAAGGCAGGACCGUGCUGGUCGUGGGCGAGAAGCUGAGCGUGCCCGGCAAGAUGGUGGACUGGCUGAGCGACAGGCCCGAGGCCACCUUCCGGGCCAGGCUGGACCUCGGCAUCCCCGGCGACGUGCCCAAGUACGACAUCAUCUUCGUGAACGUCAGGACCCCAUACAAGUACCACCAUUACCAGCAGUGCGAGGACCACGCCAUCAAGCUGAGCAUGCUGACCAAGAAGGCCUGCCUGCACCUGAACCCCGGAGGCACCUGCGUGAGCAUCGGCUACGGCUACGCCGACAGGGCCAGCGAGAGCAUCAUUGGCGCCAUCGCCAGGCUGUUCAAGUUCAGCAGGGUGUGCAAACCCAAGAGCAGCCUGGAGGAAACCGAGGUGCUGUUCGUGUUCAUCGGCUACGACCGGAAGGCCAGGACCCACAACCCCUACAAGCUGAGCAGCACCCUGACAAACAUCUACACCGGCAGCAGGCUGCACGAGGCCGGCUGCGCCCCCAGCUACCACGUGGUCAGGGGCGAUAUCGCCACCGCCACCGAGGGCGUGAUCAUCAACGCUGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGUGUGCGGCGCCCUGUACAAGAAGUUCCCCGAGAGCUUCGACCUGCAGCCCAUCGAGGUGGGCAAGGCCAGGCUGGUGAAGGGCGCCGCUAAGCACAUCAUCCACGCCGUGGGCCCCAACUUCAACAAGGUGAGCGAGGUGGAAGGCGACAAGCAGCUGGCCGAAGCCUACGAGAGCAUCGCCAAGAUCGUGAACGACAAUAACUACAAGAGCGUGGCCAUCCCACUGCUCAGCACCGGCAUCUUCAGCGGCAACAAGGACAGGCUGACCCAGAGCCUGAACCACCUGCUCACCGCCCUGGACACCACCGAUGCCGACGUGGCCAUCUACUGCAGGGACAAGAAGUGGGAGAUGACCCUGAAGGAGGCCGUGGCCAGGCGGGAGGCCGUGGAAGAGAUCUGCAUCAGCGACGACUCCAGCGUGACCGAGCCCGACGCCGAGCUGGUGAGGGUGCACCCCAAGAGCUCCCUGGCCGGCAGGAAGGGCUACAGCACCAGCGACGGCAAGACCUUCAGCUACCUGGAGGGCACCAAGUUCCACCAGGCCGCUAAGGACAUCGCCGAGAUCAACGCUAUGUGGCCCGUGGCCACCGAGGCCAACGAGCAGGUGUGCAUGUACAUCCUGGGCGAGAGCAUGUCCAGCAUCAGGAGCAAGUGCCCCGUGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCUGCCCUGCCUGUGCAUCCACGCUAUGACACCCGAGAGGGUGCAGCGGCUGAAGGCCAGCAGGCCCGAGCAGAUCACCGUGUGCAGCUCCUUCCCACUGCCCAAGUACAGGAUCACCGGCGUGCAGAAGAUCCAGUGCAGCCAGCCCAUCCUGUUCAGCCCAAAGGUGCCCGCCUACAUCCACCCCAGGAAGUACCUGGUGGAGACCCCACCCGUGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCUGAUCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCAUCAUUAUCGAGGAAGAGGAAGAGGACAGCAUCAGCCUGCUGAGCGACGGCCCCACCCACCAGGUGCUGCAGGUGGAGGCCGACAUCCACGGCCCACCCAGCGUGUCCAGCUCCAGCUGGAGCAUCCCACACGCCAGCGACUUCGACGUGGACAGCCUGAGCAUCCUGGACACCCUGGAGGGCGCCAGCGUGACCUCCGGCGCCACCAGCGCCGAGACCAACAGCUACUUCGCCAAGAGCAUGGAGUUCCUGGCCAGGCCCGUGCCAGCUCCCAGGACCGUGUUCAGGAACCCACCCCACCCAGCUCCCAGGACCAGGACCCCAAGCCUGGCUCCCAGCAGGGCCUGCAGCAGGACCAGCCUGGUGAGCACCCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAACUGGAGGCCCUGACACCCAGCAGGACCCCCAGCAGGUCCGUGAGCAGGACUAGUCUGGUGUCCAACCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAAUUCGAGGCCUUCGUGGCCCAGCAACAGAGACGGUUCGACGCCGGCGCCUACAUCUUCAGCAGCGACACCGGCCAGGGACACCUGCAGCAAAAGAGCGUGAGGCAGACCGUGCUGAGCGAGGUGGUGCUGGAGAGGACCGAGCUGGAAAUCAGCUACGCCCCCAGGCUGGACCAGGAGAAGGAGGAACUGCUCAGGAAGAAACUGCAGCUGAACCCCACCCCAGCCAACAGGAGCAGGUACCAGAGCAGGAAGGUGGAGAACAUGAAGGCCAUCACCGCCAGGCGGAUCCUGCAGGGCCUGGGACACUACCUGAAGGCCGAGGGCAAGGUGGAGUGCUACAGGACCCUGCACCCCGUGCCACUGUACAGCUCCAGCGUGAACAGGGCCUUCUCCAGCCCCAAGGUGGCCGUGGAGGCCUGCAACGCUAUGCUGAAGGAGAACUUCCCCACCGUGGCCAGCUACUGCAUCAUCCCCGAGUACGACGCCUACCUGGACAUGGUGGACGGCGCCAGCUGCUGCCUGGACACCGCCAGCUUCUGCCCCGCCAAGCUGAGGAGCUUCCCCAAGAAACACAGCUACCUGGAGCCCACCAUCAGGAGCGCCGUGCCCAGCGCCAUCCAGAACACCCUGCAGAACGUGCUGGCCGCUGCCACCAAGAGGAACUGCAACGUGACCCAGAUGAGGGAGCUGCCCGUGCUGGACAGCGCUGCCUUCAACGUGGAGUGCUUCAAGAAAUACGCCUGCAACAACGAGUACUGGGAGACCUUCAAGGAGAACCCCAUCAGGCUGACCGAAGAGAACGUGGUGAACUACAUCACCAAGCUGAAGGGCCCCAAGGCCGCUGCCCUGUUCGCUAAGACCCACAACCUGAACAUGCUGCAGGACAUCCCAAUGGACAGGUUCGUGAUGGACCUGAAGAGGGACGUGAAGGUGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGUGCAGGUGAUCCAGGCCGCUGACCCACUGGCCACCGCCUACCUGUGCGGCAUCCACAGGGAGCUGGUGAGGCGGCUGAACGCCGUGCUGCUGCCCAACAUCCACACCCUGUUCGACAUGAGCGCCGAGGACUUCGACGCCAUCAUCGCCGAGCACUUCCAGCCCGGCGACUGCGUGCUGGAGACCGACAUCGCCAGCUUCGACAAGAGCGAGGAUGACGCUAUGGCCCUGACCGCUCUGAUGAUCCUGGAGGACCUGGGCGUGGACGCCGAGCUGCUCACCCUGAUCGAGGCUGCCUUCGGCGAGAUCAGCUCCAUCCACCUGCCCACCAAGACCAAGUUCAAGUUCGGCGCUAUGAUGAAAAGCGGAAUGUUCCUGACCCUGUUCGUGAACACCGUGAUCAACAUUGUGAUCGCCAGCAGGGUGCUGCGGGAGAGGCUGACCGGCAGCCCCUGCGCUGCCUUCAUCGGCGACGACAACAUCGUGAAGGGCGUGAAAAGCGACAAGCUGAUGGCCGACAGGUGCGCCACCUGGCUGAACAUGGAGGUGAAGAUCAUCGACGCCGUGGUGGGCGAGAAGGCCCCCUACUUCUGCGGCGGAUUCAUCCUGUGCGACAGCGUGACCGGCACCGCCUGCAGGGUGGCCGACCCCCUGAAGAGGCUGUUCAAGCUGGGCAAGCCACUGGCCGCUGACGAUGAGCACGACGAUGACAGGCGGAGGGCCCUGCACGAGGAAAGCACCAGGUGGAACAGGGUGGGCAUCCUGAGCGAGCUGUGCAAGGCCGUGGAGAGCAGGUACGAGACCGUGGGCACCAGCAUCAUCGUGAUGGCUAUGACCACACUGGCCAGCUCCGUCAAGAGCUUCUCCUACCUGAGGGGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGGCCGCCACCCAUGAAGUUGGUGGUUGUGGGGGCCGGGGGUGUUGGCAAAAGCGCCCUUACAAUUUGACUCGAGUAUGUUACGUGCAAAGGUGAUUGUCACCCCCCGAAAGACCAUAUUGUGACACACCCUCAGUAUCACGCCCAAACAUUUACAGCCGCGGUGUCAAAAACCGCGUGGACGUGGUUAACAUCCCUGCUGGGAGGAUCAGCCGUAAUUAUUAUAAUUGGCUUGGUGCUGGCUACUAUUGUGGCCAUGUACGUGCUGACCAACCAGAAACAUAAUUGAAUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 2862 SINV 2862AUUGACGGCGUAGUACACACUAUUGAAUCAAACAGCCGACCAAUUG (SEQ emptyCACUACCAUCACAAUGGAGAAGCCAGUAGUAAACGUAGACGUAGAC IDCCCCAGAGUCCGUUUGUCGUGCAACUGCAAAAAAGCUUCCCGCAAU NO:104)UUGAGGUAGUAGCACAGCAGGUCACUCCAAAUGACCAUGCUAAUGCCAGAGCAUUUUCGCAUCUGGCCAGUAAACUAAUCGAGCUGGAGGUUCCUACCACAGCGACGAUCUUGGACAUAGGCAGCGCACCGGCUCGUAGAAUGUUUUCCGAGCACCAGUAUCAUUGUGUCUGCCCCAUGCGUAGUCCAGAAGACCCGGACCGCAUGAUGAAAUAUGCCAGUAAACUGGCGGAAAAAGCGUGCAAGAUUACAAACAAGAACUUGCAUGAGAAGAUUAAGGAUCUCCGGACCGUACUUGAUACGCCGGAUGCUGAAACACCAUCGCUCUGCUUUCACAACGAUGUUACCUGCAACAUGCGUGCCGAAUAUUCCGUCAUGCAGGACGUGUAUAUCAACGCUCCCGGAACUAUCUAUCAUCAGGCUAUGAAAGGCGUGCGGACCCUGUACUGGAUUGGCUUCGACACCACCCAGUUCAUGUUCUCGGCUAUGGCAGGUUCGUACCCUGCGUACAACACCAACUGGGCCGACGAGAAAGUCCUUGAAGCGCGUAACAUCGGACUUUGCAGCACAAAGCUGAGUGAAGGUAGGACAGGAAAAUUGUCGAUAAUGAGGAAGAAGGAGUUGAAGCCCGGGUCGCGGGUUUAUUUCUCCGUAGGAUCGACACUUUAUCCAGAACACAGAGCCAGCUUGCAGAGCUGGCAUCUUCCAUCGGUGUUCCACUUGAAUGGAAAGCAGUCGUACACUUGCCGCUGUGAUACAGUGGUGAGUUGCGAAGGCUACGUAGUGAAGAAAAUCACCAUCAGUCCCGGGAUCACGGGAGAAACCGUGGGAUACGCGGUUACACACAAUAGCGAGGGCUUCUUGCUAUGCAAAGUUACUGACACAGUAAAAGGAGAACGGGUAUCGUUCCCUGUGUGCACGUACAUCCCGGCCACCAUAUGCGAUCAGAUGACUGGUAUAAUGGCCACGGAUAUAUCACCUGACGAUGCACAAAAACUUCUGGUUGGGCUCAACCAGCGAAUUGUCAUUAACGGUAGGACUAACAGGAACACCAACACCAUGCAAAAUUACCUUCUGCCGAUCAUAGCACAAGGGUUCAGCAAAUGGGCUAAGGAGCGCAAGGAUGAUCUUGAUAACGAGAAAAUGCUGGGUACUAGAGAACGCAAGCUUACGUAUGGCUGCUUGUGGGCGUUUCGCACUAAGAAAGUACAUUCGUUUUAUCGCCCACCUGGAACGCAGACCUGCGUAAAAGUCCCAGCCUCUUUUAGCGCUUUUCCCAUGUCGUCCGUAUGGACGACCUCUUUGCCCAUGUCGCUGAGGCAGAAAUUGAAACUGGCAUUGCAACCAAAGAAGGAGGAAAAACUGCUGCAGGUCUCGGAGGAAUUAGUCAUGGAGGCCAAGGCUGCUUUUGAGGAUGCUCAGGAGGAAGCCAGAGCGGAGAAGCUCCGAGAAGCACUUCCACCAUUAGUGGCAGACAAAGGCAUCGAGGCAGCCGCAGAAGUUGUCUGCGAAGUGGAGGGGCUCCAGGCGGACAUCGGAGCAGCAUUAGUUGAAACCCCGCGCGGUCACGUAAGGAUAAUACCUCAAGCAAAUGACCGUAUGAUCGGACAGUAUAUCGUUGUCUCGCCAAACUCUGUGCUGAAGAAUGCCAAACUCGCACCAGCGCACCCGCUAGCAGAUCAGGUUAAGAUCAUAACACACUCCGGAAGAUCAGGAAGGUACGCGGUCGAACCAUACGACGCUAAAGUACUGAUGCCAGCAGGAGGUGCCGUACCAUGGCCAGAAUUCCUAGCACUGAGUGAGAGCGCCACGUUAGUGUACAACGAAAGAGAGUUUGUGAACCGCAAACUAUACCACAUUGCCAUGCAUGGCCCCGCCAAGAAUACAGAAGAGGAGCAGUACAAGGUUACAAAGGCAGAGCUUGCAGAAACAGAGUACGUGUUUGACGUGGACAAGAAGCGUUGCGUUAAGAAGGAAGAAGCCUCAGGUCUGGUCCUCUCGGGAGAACUGACCAACCCUCCCUAUCAUGAGCUAGCUCUGGAGGGACUGAAGACCCGACCUGCGGUCCCGUACAAGGUCGAAACAAUAGGAGUGAUAGGCACACCGGGGUCGGGCAAGUCAGCUAUUAUCAAGUCAACUGUCACGGCACGAGAUCUUGUUACCAGCGGAAAGAAAGAAAAUUGUCGCGAAAUUGAGGCCGACGUGCUAAGACUGAGGGGUAUGCAGAUUACGUCGAAGACAGUAGAUUCGGUUAUGCUCAACGGAUGCCACAAAGCCGUAGAAGUGCUGUACGUUGACGAAGCGUUCGCGUGCCACGCAGGAGCACUACUUGCCUUGAUUGCUAUCGUCAGGCCCCGCAAGAAGGUAGUACUAUGCGGAGACCCCAUGCAAUGCGGAUUCUUCAACAUGAUGCAACUAAAGGUACAUUUCAAUCACCCUGAAAAAGACAUAUGCACCAAGACAUUCUACAAGUAUAUCUCCCGGCGUUGCACACAGCCAGUUACAGCUAUUGUAUCGACACUGCAUUACGAUGGAAAGAUGAAAACCACGAACCCGUGCAAGAAGAACAUUGAAAUCGAUAUUACAGGGGCCACAAAGCCGAAGCCAGGGGAUAUCAUCCUGACAUGUUUCCGCGGGUGGGUUAAGCAAUUGCAAAUCGACUAUCCCGGACAUGAAGUAAUGACAGCCGCGGCCUCACAAGGGCUAACCAGAAAAGGAGUGUAUGCCGUCCGGCAAAAAGUCAAUGAAAACCCACUGUACGCGAUCACAUCAGAGCAUGUGAACGUGUUGCUCACCCGCACUGAGGACAGGCUAGUGUGGAAAACCUUGCAGGGCGACCCAUGGAUUAAGCAGCUCACUAACAUACCUAAAGGAAACUUUCAGGCUACUAUAGAGGACUGGGAAGCUGAACACAAGGGAAUAAUUGCUGCAAUAAACAGCCCCACUCCCCGUGCCAAUCCGUUCAGCUGCAAGACCAACGUUUGCUGGGCGAAAGCAUUGGAACCGAUACUAGCCACGGCCGGUAUCGUACUUACCGGUUGCCAGUGGAGCGAACUGUUCCCACAGUUUGCGGAUGACAAACCACAUUCGGCCAUUUACGCCUUAGACGUAAUUUGCAUUAAGUUUUUCGGCAUGGACUUGACAAGCGGACUGUUUUCUAAACAGAGCAUCCCACUAACGUACCAUCCCGCCGAUUCAGCGAGGCCGGUAGCUCAUUGGGACAACAGCCCAGGAACCCGCAAGUAUGGGUACGAUCACGCCAUUGCCGCCGAACUCUCCCGUAGAUUUCCGGUGUUCCAGCUAGCUGGGAAGGGCACACAACUUGAUUUGCAGACGGGGAGAACCAGAGUUAUCUCUGCACAGCAUAACCUGGUCCCGGUGAACCGCAAUCUUCCUCACGCCUUAGUCCCCGAGUACAAGGAGAAGCAACCCGGCCCGGUCGAAAAAUUCUUGAACCAGUUCAAACACCACUCAGUACUUGUGGUAUCAGAGGAAAAAAUUGAAGCUCCCCGUAAGAGAAUCGAAUGGAUCGCCCCGAUUGGCAUAGCCGGUGCAGAUAAGAACUACAACCUGGCUUUCGGGUUUCCGCCGCAGGCACGGUACGACCUGGUGUUCAUCAACAUUGGAACUAAAUACAGAAACCACCACUUUCAGCAGUGCGAAGACCAUGCGGCGACCUUAAAAACCCUUUCGCGUUCGGCCCUGAAUUGCCUUAACCCAGGAGGCACCCUCGUGGUGAAGUCCUAUGGCUACGCCGACCGCAACAGUGAGGACGUAGUCACCGCUCUUGCCAGAAAGUUUGUCAGGGUGUCUGCAGCGAGACCAGAUUGUGUCUCAAGCAAUACAGAAAUGUACCUGAUUUUCCGACAACUAGACAACAGCCGUACACGGCAAUUCACCCCGCACCAUCUGAAUUGCGUGAUUUCGUCCGUGUAUGAGGGUACAAGAGAUGGAGUUGGAGCCGCGCCGUCAUACCGCACCAAAAGGGAGAAUAUUGCUGACUGUCAAGAGGAAGCAGUUGUCAACGCAGCCAAUCCGCUGGGUAGACCAGGCGAAGGAGUCUGCCGUGCCAUCUAUAAACGUUGGCCGACCAGUUUUACCGAUUCAGCCACGGAGACAGGCACCGCAAGAAUGACUGUGUGCCUAGGAAAGAAAGUGAUCCACGCGGUCGGCCCUGAUUUCCGGAAGCACCCAGAAGCAGAAGCCUUGAAAUUGCUACAAAACGCCUACCAUGCAGUGGCAGACUUAGUAAAUGAACAUAACAUCAAGUCUGUCGCCAUUCCACUGCUAUCUACAGGCAUUUACGCAGCCGGAAAAGACCGCCUUGAAGUAUCACUUAACUGCUUGACAACCGCGCUAGACAGAACUGACGCGGACGUAACCAUCUAUUGCCUGGAUAAGAAGUGGAAGGAAAGAAUCGACGCGGCACUCCAACUUAAGGAGUCUGUAACAGAGCUGAAGGAUGAAGAUAUGGAGAUCGACGAUGAGUUAGUAUGGAUCCAUCCAGACAGUUGCUUGAAGGGAAGAAAGGGAUUCAGUACUACAAAAGGAAAAUUGUAUUCGUACUUCGAAGGCACCAAAUUCCAUCAAGCAGCAAAAGACAUGGCGGAGAUAAAGGUCCUGUUCCCUAAUGACCAGGAAAGUAAUGAACAACUGUGUGCCUACAUAUUGGGUGAGACCAUGGAAGCAAUCCGCGAAAAGUGCCCGGUCGACCAUAACCCGUCGUCUAGCCCGCCCAAAACGUUGCCGUGCCUUUGCAUGUAUGCCAUGACGCCAGAAAGGGUCCACAGACUUAGAAGCAAUAACGUCAAAGAAGUUACAGUAUGCUCCUCCACCCCCCUUCCUAAGCACAAAAUUAAGAAUGUUCAGAAGGUUCAGUGCACGAAAGUAGUCCUGUUUAAUCCGCACACUCCCGCAUUCGUUCCCGCCCGUAAGUACAUAGAAGUGCCAGAACAGCCUACCGCUCCUCCUGCACAGGCCGAGGAGGCCCCCGAAGUUGUAGCGACACCGUCACCAUCUACAGCUGAUAACACCUCGCUUGAUGUCACAGACAUCUCACUGGAUAUGGAUGACAGUAGCGAAGGCUCACUUUUUUCGAGCUUUAGCGGAUCGGACAACUCUAUUACUAGUAUGGACAGUUGGUCGUCAGGACCUAGUUCACUAGAGAUAGUAGACCGAAGGCAGGUGGUGGUGGCUGACGUUCAUGCCGUCCAAGAGCCUGCCCCUAUUCCACCGCCAAGGCUAAAGAAGAUGGCCCGCCUGGCAGCGGCAAGAAAAGAGCCCACUCCACCGGCAAGCAAUAGCUCUGAGUCCCUCCACCUCUCUUUUGGUGGGGUAUCCAUGUCCCUCGGAUCAAUUUUCGACGGAGAGACGGCCCGCCAGGCAGCGGUACAACCCCUGGCAACAGGCCCCACGGAUGUGCCUAUGUCUUUCGGAUCGUUUUCCGACGGAGAGAUUGAUGAGCUGAGCCGCAGAGUAACUGAGUCCGAACCCGUCCUGUUUGGAUCAUUUGAACCGGGCGAAGUGAACUCAAUUAUAUCGUCCCGAUCAGCCGUAUCUUUUCCUCUACGCAAGCAGAGACGUAGACGCAGGAGCAGGAGGACUGAAUACUGACUAACCGGGGUAGGUGGGUACAUAUUUUCGACGGACACAGGCCCUGGGCACUUGCAAAAGAAGUCCGUUCUGCAGAACCAGCUUACAGAACCGACCUUGGAGCGCAAUGUCCUGGAAAGAAUUCAUGCCCCGGUGCUCGACACGUCGAAAGAGGAACAACUCAAACUCAGGUACCAGAUGAUGCCCACCGAAGCCAACAAAAGUAGGUACCAGUCUCGUAAAGUAGAAAAUCAGAAAGCCAUAACCACUGAGCGACUACUGUCAGGACUACGACUGUAUAACUCUGCCACAGAUCAGCCAGAAUGCUAUAAGAUCACCUAUCCGAAACCAUUGUACUCCAGUAGCGUACCGGCGAACUACUCCGAUCCACAGUUCGCUGUAGCUGUCUGUAACAACUAUCUGCAUGAGAACUAUCCGACAGUAGCAUCUUAUCAGAUUACUGACGAGUACGAUGCUUACUUGGAUAUGGUAGACGGGACAGUCGCCUGCCUGGACACUGCAACCUUCUGCCCCGCUAAGCUUAGAAGUUACCCGAAAAAACAUGAGUAUAGAGCCCCGAAUAUCCGCAGUGCGGUUCCAUCAGCGAUGCAGAACACGCUACAAAAUGUGCUCAUUGCCGCAACUAAAAGAAAUUGCAACGUCACGCAGAUGCGUGAACUGCCAACACUGGACUCAGCGACAUUCAAUGUCGAAUGCUUUCGAAAAUAUGCAUGUAAUGACGAGUAUUGGGAGGAGUUCGCUCGGAAGCCAAUUAGGAUUACCACUGAGUUUGUCACCGCAUAUGUAGCUAGACUGAAAGGCCCUAAGGCCGCCGCACUAUUUGCAAAGACGUAUAAUUUGGUCCCAUUGCAAGAAGUGCCUAUGGAUAGAUUCGUCAUGGACAUGAAAAGAGACGUGAAAGUUACACCAGGCACGAAACACACAGAAGAAAGACCGAAAGUACAAGUGAUACAAGCCGCAGAACCCCUGGCGACUGCUUACUUAUGCGGGAUUCACCGGGAAUUAGUGCGUAGGCUUACGGCCGUCUUGCUUCCAAACAUUCACACGCUUUUUGACAUGUCGGCGGAGGAUUUUGAUGCAAUCAUAGCAGAACACUUCAAGCAAGGCGACCCGGUACUGGAGACGGAUAUCGCAUCAUUCGACAAAAGCCAAGACGACGCUAUGGCGUUAACCGGUCUGAUGAUCUUGGAGGACCUGGGUGUGGAUCAACCACUACUCGACUUGAUCGAGUGCGCCUUUGGAGAAAUAUCAUCCACCCAUCUACCUACGGGUACUCGUUUUAAAUUCGGGGCGAUGAUGAAAUCCGGAAUGUUCCUCACACUUUUUGUCAACACAGUUUUGAAUGUCGUUAUCGCCAGCAGAGUACUAGAGGAGCGGCUUAAAACGUCCAGAUGUGCAGCGUUCAUUGGCGACGACAACAUCAUACAUGGAGUAGUAUCUGACAAAGAAAUGGCUGAGAGGUGCGCCACCUGGCUCAACAUGGAGGUUAAGAUCAUCGACGCAGUCAUCGGUGAGAGACCACCUUACUUCUGCGGCGGAUUUAUCUUGCAAGAUUCGGUUACUUCCACAGCGUGCCGCGUGGCGGAUCCCCUGAAAAGGCUGUUUAAGUUGGGUAAACCGCUCCCAGCCGACGACGAGCAAGACGAAGACAGAAGACGCGCUCUGCUAGAUGAAACAAAGGCGUGGUUUAGAGUAGGUAUAACAGGCACUUUAGCAGUGGCCGUGACGACCCGGUAUGAGGUAGACAAUAUUACACCUGUCCUACUGGCAUUGAGAACUUUUGCCCAGAGCAAAAGAGCAUUCCAAGCCAUCAGAGGGGAAAUAAAGCAUCUCUACGGUGGUCCUAAAUAGUCAGCAUAGUACAUUUCAUCUGACUAAUACUACAACACCACCACCACGCGUGCUAGACCAUGGAUCCUAGACGCUACGCCCCAAUGAUCCGACCAGCAAAACUCGAUGUACUUCCGAGGAACUGAUGUGCAUAAUGCAUCAGGCUGGUACAUUAGAUCCCCGCUUACCGCGGGCAAUAUAGCAACACUAAAAACUCGAUGUACUUCCGAGGAAGCGCAGUGCAUAAUGCUGCGCAGUGUUGCCACAUAACCACUAUAUUAACCAUUUAUCUAGCGGACGCCAAAAACUCAAUGUAUUUCUGAGGAAGCGUGGUGCAUAAUGCCACGCAGCGUCUGCAUAACUUUUAUUAUUUCUUUUAUUAAUCAACAAAAUUUUGUUUUUAACAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA3060 STARR^(TM) 3060 AUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAAAU (SEQgp70 GGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGA IDGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGG NO:105)UCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCCGUCGACGGCCCCACCAGCCUGUACCACCAGGCCAACAAGGGCGUGAGGGUGGCCUACUGGAUCGGCUUCGACACCACACCCUUCAUGUUCAAGAACCUGGCCGGCGCCUACCCCAGCUACAGCACCAACUGGGCCGACGAGACCGUGCUGACCGCCAGGAACAUCGGCCUGUGCAGCAGCGACGUGAUGGAGAGGAGCCGGAGAGGCAUGAGCAUCCUGAGGAAGAAAUACCUGAAGCCCAGCAACAACGUGCUGUUCAGCGUGGGCAGCACCAUCUACCACGAGAAGAGGGACCUGCUCAGGAGCUGGCACCUGCCCAGCGUGUUCCACCUGAGGGGCAAGCAGAACUACACCUGCAGGUGCGAGACCAUCGUGAGCUGCGACGGCUACGUGGUGAAGAGGAUCGCCAUCAGCCCCGGCCUGUACGGCAAGCCCAGCGGCUACGCCGCUACAAUGCACAGGGAGGGCUUCCUGUGCUGCAAGGUGACCGACACCCUGAACGGCGAGAGGGUGAGCUUCCCCGUGUGCACCUACGUGCCCGCCACCCUGUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGAGCGCCGACGACGCCCAGAAGCUGCUCGUGGGCCUGAACCAGAGGAUCGUGGUCAACGGCAGGACCCAGAGGAACACCAACACAAUGAAGAACUACCUGCUGCCCGUGGUGGCCCAGGCUUUCGCCAGGUGGGCCAAGGAGUACAAGGAGGACCAGGAAGACGAGAGGCCCCUGGGCCUGAGGGACAGGCAGCUGGUGAUGGGCUGCUGCUGGGCCUUCAGGCGGCACAAGAUCACCAGCAUCUACAAGAGGCCCGACACCCAGACCAUCAUCAAGGUGAACAGCGACUUCCACAGCUUCGUGCUGCCCAGGAUCGGCAGCAACACCCUGGAGAUCGGCCUGAGGACCCGGAUCAGGAAGAUGCUGGAGGAACACAAGGAGCCCAGCCCACUGAUCACCGCCGAGGACGUGCAGGAGGCCAAGUGCGCUGCCGACGAGGCCAAGGAGGUGAGGGAGGCCGAGGAACUGAGGGCCGCCCUGCCACCCCUGGCUGCCGACGUGGAGGAACCCACCCUGGAAGCCGACGUGGACCUGAUGCUGCAGGAGGCCGGCGCCGGAAGCGUGGAGACACCCAGGGGCCUGAUCAAGGUGACCAGCUACGACGGCGAGGACAAGAUCGGCAGCUACGCCGUGCUGAGCCCACAGGCCGUGCUGAAGUCCGAGAAGCUGAGCUGCAUCCACCCACUGGCCGAGCAGGUGAUCGUGAUCACCCACAGCGGCAGGAAGGGCAGGUACGCCGUGGAGCCCUACCACGGCAAGGUGGUCGUGCCCGAGGGCCACGCCAUCCCCGUGCAGGACUUCCAGGCCCUGAGCGAGAGCGCCACCAUCGUGUACAACGAGAGGGAGUUCGUGAACAGGUACCUGCACCAUAUCGCCACCCACGGCGGAGCCCUGAACACCGACGAGGAAUACUACAAGACCGUGAAGCCCAGCGAGCACGACGGCGAGUACCUGUACGACAUCGACAGGAAGCAGUGCGUGAAGAAAGAGCUGGUGACCGGCCUGGGACUGACCGGCGAGCUGGUGGACCCACCCUUCCACGAGUUCGCCUACGAGAGCCUGAGGACCAGACCCGCCGCUCCCUACCAGGUGCCCACCAUCGGCGUGUACGGCGUGCCCGGCAGCGGAAAGAGCGGCAUCAUCAAGAGCGCCGUGACCAAGAAAGACCUGGUGGUCAGCGCCAAGAAAGAGAACUGCGCCGAGAUCAUCAGGGACGUGAAGAAGAUGAAAGGCCUGGACGUGAACGCGCGCACCGUGGACAGCGUGCUGCUGAACGGCUGCAAGCACCCCGUGGAGACCCUGUACAUCGACGAGGCCUUCGCUUGCCACGCCGGCACCCUGAGGGCCCUGAUCGCCAUCAUCAGGCCCAAGAAAGCCGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUGAUGUGCCUGAAGGUGCACUUCAACCACGAGAUCUGCACCCAGGUGUUCCACAAGAGCAUCAGCAGGCGGUGCACCAAGAGCGUGACCAGCGUCGUGAGCACCCUGUUCUACGACAAGAAAAUGAGGACCACCAACCCCAAGGAGACCAAAAUCGUGAUCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCUGAUCCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCAGAUCGACUACAAGGGCAACGAGAUCAUGACCGCCGCUGCCAGCCAGGGCCUGACCAGGAAGGGCGUGUACGCCGUGAGGUACAAGGUGAACGAGAACCCACUGUACGCUCCCACCAGCGAGCACGUGAACGUGCUGCUGACCAGGACCGAGGACAGGAUCGUGUGGAAGACCCUGGCCGGCGACCCCUGGAUCAAGACCCUGACCGCCAAGUACCCCGGCAACUUCACCGCCACCAUCGAAGAGUGGCAGGCCGAGCACGACGCCAUCAUGAGGCACAUCCUGGAGAGGCCCGACCCCACCGACGUGUUCCAGAACAAGGCCAACGUGUGCUGGGCCAAGGCCCUGGUGCCCGUGCUGAAGACCGCCGGCAUCGACAUGACCACAGAGCAGUGGAACACCGUGGACUACUUCGAGACCGACAAGGCCCACAGCGCCGAGAUCGUGCUGAACCAGCUGUGCGUGAGGUUCUUCGGCCUGGACCUGGACAGCGGCCUGUUCAGCGCCCCCACCGUGCCACUGAGCAUCAGGAACAACCACUGGGACAACAGCCCCAGCCCAAACAUGUACGGCCUGAACAAGGAGGUGGUCAGGCAGCUGAGCAGGCGGUACCCACAGCUGCCCAGGGCCGUGGCCACCGGCAGGGUGUACGACAUGAACACCGGCACCCUGAGGAACUACGACCCCAGGAUCAACCUGGUGCCCGUGAACAGGCGGCUGCCCCACGCCCUGGUGCUGCACCACAACGAGCACCCACAGAGCGACUUCAGCUCCUUCGUGAGCAAGCUGAAAGGCAGGACCGUGCUGGUCGUGGGCGAGAAGCUGAGCGUGCCCGGCAAGAUGGUGGACUGGCUGAGCGACAGGCCCGAGGCCACCUUCCGGGCCAGGCUGGACCUCGGCAUCCCCGGCGACGUGCCCAAGUACGACAUCAUCUUCGUGAACGUCAGGACCCCAUACAAGUACCACCAUUACCAGCAGUGCGAGGACCACGCCAUCAAGCUGAGCAUGCUGACCAAGAAGGCCUGCCUGCACCUGAACCCCGGAGGCACCUGCGUGAGCAUCGGCUACGGCUACGCCGACAGGGCCAGCGAGAGCAUCAUUGGCGCCAUCGCCAGGCUGUUCAAGUUCAGCAGGGUGUGCAAACCCAAGAGCAGCCUGGAGGAAACCGAGGUGCUGUUCGUGUUCAUCGGCUACGACCGGAAGGCCAGGACCCACAACCCCUACAAGCUGAGCAGCACCCUGACAAACAUCUACACCGGCAGCAGGCUGCACGAGGCCGGCUGCGCCCCCAGCUACCACGUGGUCAGGGGCGAUAUCGCCACCGCCACCGAGGGCGUGAUCAUCAACGCUGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGUGUGCGGCGCCCUGUACAAGAAGUUCCCCGAGAGCUUCGACCUGCAGCCCAUCGAGGUGGGCAAGGCCAGGCUGGUGAAGGGCGCCGCUAAGCACAUCAUCCACGCCGUGGGCCCCAACUUCAACAAGGUGAGCGAGGUGGAAGGCGACAAGCAGCUGGCCGAAGCCUACGAGAGCAUCGCCAAGAUCGUGAACGACAAUAACUACAAGAGCGUGGCCAUCCCACUGCUCAGCACCGGCAUCUUCAGCGGCAACAAGGACAGGCUGACCCAGAGCCUGAACCACCUGCUCACCGCCCUGGACACCACCGAUGCCGACGUGGCCAUCUACUGCAGGGACAAGAAGUGGGAGAUGACCCUGAAGGAGGCCGUGGCCAGGCGGGAGGCCGUGGAAGAGAUCUGCAUCAGCGACGACUCCAGCGUGACCGAGCCCGACGCCGAGCUGGUGAGGGUGCACCCCAAGAGCUCCCUGGCCGGCAGGAAGGGCUACAGCACCAGCGACGGCAAGACCUUCAGCUACCUGGAGGGCACCAAGUUCCACCAGGCCGCUAAGGACAUCGCCGAGAUCAACGCUAUGUGGCCCGUGGCCACCGAGGCCAACGAGCAGGUGUGCAUGUACAUCCUGGGCGAGAGCAUGUCCAGCAUCAGGAGCAAGUGCCCCGUGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCUGCCCUGCCUGUGCAUCCACGCUAUGACACCCGAGAGGGUGCAGCGGCUGAAGGCCAGCAGGCCCGAGCAGAUCACCGUGUGCAGCUCCUUCCCACUGCCCAAGUACAGGAUCACCGGCGUGCAGAAGAUCCAGUGCAGCCAGCCCAUCCUGUUCAGCCCAAAGGUGCCCGCCUACAUCCACCCCAGGAAGUACCUGGUGGAGACCCCACCCGUGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCUGAUCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCAUCAUUAUCGAGGAAGAGGAAGAGGACAGCAUCAGCCUGCUGAGCGACGGCCCCACCCACCAGGUGCUGCAGGUGGAGGCCGACAUCCACGGCCCACCCAGCGUGUCCAGCUCCAGCUGGAGCAUCCCACACGCCAGCGACUUCGACGUGGACAGCCUGAGCAUCCUGGACACCCUGGAGGGCGCCAGCGUGACCUCCGGCGCCACCAGCGCCGAGACCAACAGCUACUUCGCCAAGAGCAUGGAGUUCCUGGCCAGGCCCGUGCCAGCUCCCAGGACCGUGUUCAGGAACCCACCCCACCCAGCUCCCAGGACCAGGACCCCAAGCCUGGCUCCCAGCAGGGCCUGCAGCAGGACCAGCCUGGUGAGCACCCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAACUGGAGGCCCUGACACCCAGCAGGACCCCCAGCAGGUCCGUGAGCAGGACUAGUCUGGUGUCCAACCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAAUUCGAGGCCUUCGUGGCCCAGCAACAGAGACGGUUCGACGCCGGCGCCUACAUCUUCAGCAGCGACACCGGCCAGGGACACCUGCAGCAAAAGAGCGUGAGGCAGACCGUGCUGAGCGAGGUGGUGCUGGAGAGGACCGAGCUGGAAAUCAGCUACGCCCCCAGGCUGGACCAGGAGAAGGAGGAACUGCUCAGGAAGAAACUGCAGCUGAACCCCACCCCAGCCAACAGGAGCAGGUACCAGAGCAGGAAGGUGGAGAACAUGAAGGCCAUCACCGCCAGGCGGAUCCUGCAGGGCCUGGGACACUACCUGAAGGCCGAGGGCAAGGUGGAGUGCUACAGGACCCUGCACCCCGUGCCACUGUACAGCUCCAGCGUGAACAGGGCCUUCUCCAGCCCCAAGGUGGCCGUGGAGGCCUGCAACGCUAUGCUGAAGGAGAACUUCCCCACCGUGGCCAGCUACUGCAUCAUCCCCGAGUACGACGCCUACCUGGACAUGGUGGACGGCGCCAGCUGCUGCCUGGACACCGCCAGCUUCUGCCCCGCCAAGCUGAGGAGCUUCCCCAAGAAACACAGCUACCUGGAGCCCACCAUCAGGAGCGCCGUGCCCAGCGCCAUCCAGAACACCCUGCAGAACGUGCUGGCCGCUGCCACCAAGAGGAACUGCAACGUGACCCAGAUGAGGGAGCUGCCCGUGCUGGACAGCGCUGCCUUCAACGUGGAGUGCUUCAAGAAAUACGCCUGCAACAACGAGUACUGGGAGACCUUCAAGGAGAACCCCAUCAGGCUGACCGAAGAGAACGUGGUGAACUACAUCACCAAGCUGAAGGGCCCCAAGGCCGCUGCCCUGUUCGCUAAGACCCACAACCUGAACAUGCUGCAGGACAUCCCAAUGGACAGGUUCGUGAUGGACCUGAAGAGGGACGUGAAGGUGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGUGCAGGUGAUCCAGGCCGCUGACCCACUGGCCACCGCCUACCUGUGCGGCAUCCACAGGGAGCUGGUGAGGCGGCUGAACGCCGUGCUGCUGCCCAACAUCCACACCCUGUUCGACAUGAGCGCCGAGGACUUCGACGCCAUCAUCGCCGAGCACUUCCAGCCCGGCGACUGCGUGCUGGAGACCGACAUCGCCAGCUUCGACAAGAGCGAGGAUGACGCUAUGGCCCUGACCGCUCUGAUGAUCCUGGAGGACCUGGGCGUGGACGCCGAGCUGCUCACCCUGAUCGAGGCUGCCUUCGGCGAGAUCAGCUCCAUCCACCUGCCCACCAAGACCAAGUUCAAGUUCGGCGCUAUGAUGAAAAGCGGAAUGUUCCUGACCCUGUUCGUGAACACCGUGAUCAACAUUGUGAUCGCCAGCAGGGUGCUGCGGGAGAGGCUGACCGGCAGCCCCUGCGCUGCCUUCAUCGGCGACGACAACAUCGUGAAGGGCGUGAAAAGCGACAAGCUGAUGGCCGACAGGUGCGCCACCUGGCUGAACAUGGAGGUGAAGAUCAUCGACGCCGUGGUGGGCGAGAAGGCCCCCUACUUCUGCGGCGGAUUCAUCCUGUGCGACAGCGUGACCGGCACCGCCUGCAGGGUGGCCGACCCCCUGAAGAGGCUGUUCAAGCUGGGCAAGCCACUGGCCGCUGACGAUGAGCACGACGAUGACAGGCGGAGGGCCCUGCACGAGGAAAGCACCAGGUGGAACAGGGUGGGCAUCCUGAGCGAGCUGUGCAAGGCCGUGGAGAGCAGGUACGAGACCGUGGGCACCAGCAUCAUCGUGAUGGCUAUGACCACACUGGCCAGCUCCGUCAAGAGCUUCUCCUACCUGAGGGGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGGCCGCCACCAUGAGAGUGACAGCCCCUAGAACCUUACUGCUUCUGCUUUGGGGAGCUGUUGCUCUGACAGAGACAUGGGCUGGAUCUCUGAGCGAGGUGACCGGCCAGGGCCUGUGCAUCGGCGCCGUGCCCAAGACCCACCAGGUGCUGUGCAACACCACCCAGAAGACCAGCGACGGCAGCUACUACCUGGCCGCUCCCACCGGCACCACCUGGGCCUGCAGCACCGGCCUGACCCCUUGCAUCAGCACCACCAUCCUGAACCUGACCACCGACUACUGCGUGCUGGUGGAGCUGUGGCCCAGGGUGACCUACCACAGCCCCAGCUACGCCUACCACCAGUUCGAGAGGAGGGCCAAGUACAAGAGGGAGCCCGUGAGCCUGACCCUGGCCCUGCUGCUGGGCGGCCUGACAAUGGGCGGCAUCGCCGCCGGCGUGGGCACCGGCACCACCGCCCUGGUGGCCACCCAGCAGUUCCAGCAGCUGCAGGCCGCCAUGCACGACGACCUGAAGGAGGUGGAGAAGUCCAUCACCAACCUGGAGAAGUCCCUGACCAGCCUGAGCGAGGUGGUGCUGCAGAACAGGAGGGGCCUGGACCUGCUGUUCCUGAAGGAGGGCGGCCUGUGCGCCGCCCUGAAGGAGGAGUGCUGCCUGUACGCCGACCACACCGGCCUGGUGAUCGUGGGCAUUGUCGCUGGCCUGGCCGUCCUCGCCGUGGUGGUGAUUGGAGCUGUGGUCGCAGCUGUUAUGUGCAGAAGAAAGUCAUCCGGCGGAAAGGGAGGCUCCUACUCUCAGGCUGCUUCUGCUACAGUGCCUAGAGCUCUUAUGUGUUUAUCUCAGCUGUAAACUCGAGUAUGUUACGUGCAAAGGUGAUUGUCACCCCCCGAAAGACCAUAUUGUGACACACCCUCAGUAUCACGCCCAAACAUUUACAGCCGCGGUGUCAAAAACCGCGUGGACGUGGUUAACAUCCCUGCUGGGAGGAUCAGCCGUAAUUAUUAUAAUUGGCUUGGUGCUGGCUACUAUUGUGGCCAUGUACGUGCUGACCAACCAGAAACAUAAUUGAAUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3061 STARR^(TM) 3061 AUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAA (SEQ AH1A5AUGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCU IDCAGAGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCA NO:106)AGCAGGUCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCCGUCGACGGCCCCACCAGCCUGUACCACCAGGCCAACAAGGGCGUGAGGGUGGCCUACUGGAUCGGCUUCGACACCACACCCUUCAUGUUCAAGAACCUGGCCGGCGCCUACCCCAGCUACAGCACCAACUGGGCCGACGAGACCGUGCUGACCGCCAGGAACAUCGGCCUGUGCAGCAGCGACGUGAUGGAGAGGAGCCGGAGAGGCAUGAGCAUCCUGAGGAAGAAAUACCUGAAGCCCAGCAACAACGUGCUGUUCAGCGUGGGCAGCACCAUCUACCACGAGAAGAGGGACCUGCUCAGGAGCUGGCACCUGCCCAGCGUGUUCCACCUGAGGGGCAAGCAGAACUACACCUGCAGGUGCGAGACCAUCGUGAGCUGCGACGGCUACGUGGUGAAGAGGAUCGCCAUCAGCCCCGGCCUGUACGGCAAGCCCAGCGGCUACGCCGCUACAAUGCACAGGGAGGGCUUCCUGUGCUGCAAGGUGACCGACACCCUGAACGGCGAGAGGGUGAGCUUCCCCGUGUGCACCUACGUGCCCGCCACCCUGUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGAGCGCCGACGACGCCCAGAAGCUGCUCGUGGGCCUGAACCAGAGGAUCGUGGUCAACGGCAGGACCCAGAGGAACACCAACACAAUGAAGAACUACCUGCUGCCCGUGGUGGCCCAGGCUUUCGCCAGGUGGGCCAAGGAGUACAAGGAGGACCAGGAAGACGAGAGGCCCCUGGGCCUGAGGGACAGGCAGCUGGUGAUGGGCUGCUGCUGGGCCUUCAGGCGGCACAAGAUCACCAGCAUCUACAAGAGGCCCGACACCCAGACCAUCAUCAAGGUGAACAGCGACUUCCACAGCUUCGUGCUGCCCAGGAUCGGCAGCAACACCCUGGAGAUCGGCCUGAGGACCCGGAUCAGGAAGAUGCUGGAGGAACACAAGGAGCCCAGCCCACUGAUCACCGCCGAGGACGUGCAGGAGGCCAAGUGCGCUGCCGACGAGGCCAAGGAGGUGAGGGAGGCCGAGGAACUGAGGGCCGCCCUGCCACCCCUGGCUGCCGACGUGGAGGAACCCACCCUGGAAGCCGACGUGGACCUGAUGCUGCAGGAGGCCGGCGCCGGAAGCGUGGAGACACCCAGGGGCCUGAUCAAGGUGACCAGCUACGACGGCGAGGACAAGAUCGGCAGCUACGCCGUGCUGAGCCCACAGGCCGUGCUGAAGUCCGAGAAGCUGAGCUGCAUCCACCCACUGGCCGAGCAGGUGAUCGUGAUCACCCACAGCGGCAGGAAGGGCAGGUACGCCGUGGAGCCCUACCACGGCAAGGUGGUCGUGCCCGAGGGCCACGCCAUCCCCGUGCAGGACUUCCAGGCCCUGAGCGAGAGCGCCACCAUCGUGUACAACGAGAGGGAGUUCGUGAACAGGUACCUGCACCAUAUCGCCACCCACGGCGGAGCCCUGAACACCGACGAGGAAUACUACAAGACCGUGAAGCCCAGCGAGCACGACGGCGAGUACCUGUACGACAUCGACAGGAAGCAGUGCGUGAAGAAAGAGCUGGUGACCGGCCUGGGACUGACCGGCGAGCUGGUGGACCCACCCUUCCACGAGUUCGCCUACGAGAGCCUGAGGACCAGACCCGCCGCUCCCUACCAGGUGCCCACCAUCGGCGUGUACGGCGUGCCCGGCAGCGGAAAGAGCGGCAUCAUCAAGAGCGCCGUGACCAAGAAAGACCUGGUGGUCAGCGCCAAGAAAGAGAACUGCGCCGAGAUCAUCAGGGACGUGAAGAAGAUGAAAGGCCUGGACGUGAACGCGCGCACCGUGGACAGCGUGCUGCUGAACGGCUGCAAGCACCCCGUGGAGACCCUGUACAUCGACGAGGCCUUCGCUUGCCACGCCGGCACCCUGAGGGCCCUGAUCGCCAUCAUCAGGCCCAAGAAAGCCGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUGAUGUGCCUGAAGGUGCACUUCAACCACGAGAUCUGCACCCAGGUGUUCCACAAGAGCAUCAGCAGGCGGUGCACCAAGAGCGUGACCAGCGUCGUGAGCACCCUGUUCUACGACAAGAAAAUGAGGACCACCAACCCCAAGGAGACCAAAAUCGUGAUCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCUGAUCCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCAGAUCGACUACAAGGGCAACGAGAUCAUGACCGCCGCUGCCAGCCAGGGCCUGACCAGGAAGGGCGUGUACGCCGUGAGGUACAAGGUGAACGAGAACCCACUGUACGCUCCCACCAGCGAGCACGUGAACGUGCUGCUGACCAGGACCGAGGACAGGAUCGUGUGGAAGACCCUGGCCGGCGACCCCUGGAUCAAGACCCUGACCGCCAAGUACCCCGGCAACUUCACCGCCACCAUCGAAGAGUGGCAGGCCGAGCACGACGCCAUCAUGAGGCACAUCCUGGAGAGGCCCGACCCCACCGACGUGUUCCAGAACAAGGCCAACGUGUGCUGGGCCAAGGCCCUGGUGCCCGUGCUGAAGACCGCCGGCAUCGACAUGACCACAGAGCAGUGGAACACCGUGGACUACUUCGAGACCGACAAGGCCCACAGCGCCGAGAUCGUGCUGAACCAGCUGUGCGUGAGGUUCUUCGGCCUGGACCUGGACAGCGGCCUGUUCAGCGCCCCCACCGUGCCACUGAGCAUCAGGAACAACCACUGGGACAACAGCCCCAGCCCAAACAUGUACGGCCUGAACAAGGAGGUGGUCAGGCAGCUGAGCAGGCGGUACCCACAGCUGCCCAGGGCCGUGGCCACCGGCAGGGUGUACGACAUGAACACCGGCACCCUGAGGAACUACGACCCCAGGAUCAACCUGGUGCCCGUGAACAGGCGGCUGCCCCACGCCCUGGUGCUGCACCACAACGAGCACCCACAGAGCGACUUCAGCUCCUUCGUGAGCAAGCUGAAAGGCAGGACCGUGCUGGUCGUGGGCGAGAAGCUGAGCGUGCCCGGCAAGAUGGUGGACUGGCUGAGCGACAGGCCCGAGGCCACCUUCCGGGCCAGGCUGGACCUCGGCAUCCCCGGCGACGUGCCCAAGUACGACAUCAUCUUCGUGAACGUCAGGACCCCAUACAAGUACCACCAUUACCAGCAGUGCGAGGACCACGCCAUCAAGCUGAGCAUGCUGACCAAGAAGGCCUGCCUGCACCUGAACCCCGGAGGCACCUGCGUGAGCAUCGGCUACGGCUACGCCGACAGGGCCAGCGAGAGCAUCAUUGGCGCCAUCGCCAGGCUGUUCAAGUUCAGCAGGGUGUGCAAACCCAAGAGCAGCCUGGAGGAAACCGAGGUGCUGUUCGUGUUCAUCGGCUACGACCGGAAGGCCAGGACCCACAACCCCUACAAGCUGAGCAGCACCCUGACAAACAUCUACACCGGCAGCAGGCUGCACGAGGCCGGCUGCGCCCCCAGCUACCACGUGGUCAGGGGCGAUAUCGCCACCGCCACCGAGGGCGUGAUCAUCAACGCUGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGUGUGCGGCGCCCUGUACAAGAAGUUCCCCGAGAGCUUCGACCUGCAGCCCAUCGAGGUGGGCAAGGCCAGGCUGGUGAAGGGCGCCGCUAAGCACAUCAUCCACGCCGUGGGCCCCAACUUCAACAAGGUGAGCGAGGUGGAAGGCGACAAGCAGCUGGCCGAAGCCUACGAGAGCAUCGCCAAGAUCGUGAACGACAAUAACUACAAGAGCGUGGCCAUCCCACUGCUCAGCACCGGCAUCUUCAGCGGCAACAAGGACAGGCUGACCCAGAGCCUGAACCACCUGCUCACCGCCCUGGACACCACCGAUGCCGACGUGGCCAUCUACUGCAGGGACAAGAAGUGGGAGAUGACCCUGAAGGAGGCCGUGGCCAGGCGGGAGGCCGUGGAAGAGAUCUGCAUCAGCGACGACUCCAGCGUGACCGAGCCCGACGCCGAGCUGGUGAGGGUGCACCCCAAGAGCUCCCUGGCCGGCAGGAAGGGCUACAGCACCAGCGACGGCAAGACCUUCAGCUACCUGGAGGGCACCAAGUUCCACCAGGCCGCUAAGGACAUCGCCGAGAUCAACGCUAUGUGGCCCGUGGCCACCGAGGCCAACGAGCAGGUGUGCAUGUACAUCCUGGGCGAGAGCAUGUCCAGCAUCAGGAGCAAGUGCCCCGUGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCUGCCCUGCCUGUGCAUCCACGCUAUGACACCCGAGAGGGUGCAGCGGCUGAAGGCCAGCAGGCCCGAGCAGAUCACCGUGUGCAGCUCCUUCCCACUGCCCAAGUACAGGAUCACCGGCGUGCAGAAGAUCCAGUGCAGCCAGCCCAUCCUGUUCAGCCCAAAGGUGCCCGCCUACAUCCACCCCAGGAAGUACCUGGUGGAGACCCCACCCGUGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCUGAUCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCAUCAUUAUCGAGGAAGAGGAAGAGGACAGCAUCAGCCUGCUGAGCGACGGCCCCACCCACCAGGUGCUGCAGGUGGAGGCCGACAUCCACGGCCCACCCAGCGUGUCCAGCUCCAGCUGGAGCAUCCCACACGCCAGCGACUUCGACGUGGACAGCCUGAGCAUCCUGGACACCCUGGAGGGCGCCAGCGUGACCUCCGGCGCCACCAGCGCCGAGACCAACAGCUACUUCGCCAAGAGCAUGGAGUUCCUGGCCAGGCCCGUGCCAGCUCCCAGGACCGUGUUCAGGAACCCACCCCACCCAGCUCCCAGGACCAGGACCCCAAGCCUGGCUCCCAGCAGGGCCUGCAGCAGGACCAGCCUGGUGAGCACCCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAACUGGAGGCCCUGACACCCAGCAGGACCCCCAGCAGGUCCGUGAGCAGGACUAGUCUGGUGUCCAACCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAAUUCGAGGCCUUCGUGGCCCAGCAACAGAGACGGUUCGACGCCGGCGCCUACAUCUUCAGCAGCGACACCGGCCAGGGACACCUGCAGCAAAAGAGCGUGAGGCAGACCGUGCUGAGCGAGGUGGUGCUGGAGAGGACCGAGCUGGAAAUCAGCUACGCCCCCAGGCUGGACCAGGAGAAGGAGGAACUGCUCAGGAAGAAACUGCAGCUGAACCCCACCCCAGCCAACAGGAGCAGGUACCAGAGCAGGAAGGUGGAGAACAUGAAGGCCAUCACCGCCAGGCGGAUCCUGCAGGGCCUGGGACACUACCUGAAGGCCGAGGGCAAGGUGGAGUGCUACAGGACCCUGCACCCCGUGCCACUGUACAGCUCCAGCGUGAACAGGGCCUUCUCCAGCCCCAAGGUGGCCGUGGAGGCCUGCAACGCUAUGCUGAAGGAGAACUUCCCCACCGUGGCCAGCUACUGCAUCAUCCCCGAGUACGACGCCUACCUGGACAUGGUGGACGGCGCCAGCUGCUGCCUGGACACCGCCAGCUUCUGCCCCGCCAAGCUGAGGAGCUUCCCCAAGAAACACAGCUACCUGGAGCCCACCAUCAGGAGCGCCGUGCCCAGCGCCAUCCAGAACACCCUGCAGAACGUGCUGGCCGCUGCCACCAAGAGGAACUGCAACGUGACCCAGAUGAGGGAGCUGCCCGUGCUGGACAGCGCUGCCUUCAACGUGGAGUGCUUCAAGAAAUACGCCUGCAACAACGAGUACUGGGAGACCUUCAAGGAGAACCCCAUCAGGCUGACCGAAGAGAACGUGGUGAACUACAUCACCAAGCUGAAGGGCCCCAAGGCCGCUGCCCUGUUCGCUAAGACCCACAACCUGAACAUGCUGCAGGACAUCCCAAUGGACAGGUUCGUGAUGGACCUGAAGAGGGACGUGAAGGUGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGUGCAGGUGAUCCAGGCCGCUGACCCACUGGCCACCGCCUACCUGUGCGGCAUCCACAGGGAGCUGGUGAGGCGGCUGAACGCCGUGCUGCUGCCCAACAUCCACACCCUGUUCGACAUGAGCGCCGAGGACUUCGACGCCAUCAUCGCCGAGCACUUCCAGCCCGGCGACUGCGUGCUGGAGACCGACAUCGCCAGCUUCGACAAGAGCGAGGAUGACGCUAUGGCCCUGACCGCUCUGAUGAUCCUGGAGGACCUGGGCGUGGACGCCGAGCUGCUCACCCUGAUCGAGGCUGCCUUCGGCGAGAUCAGCUCCAUCCACCUGCCCACCAAGACCAAGUUCAAGUUCGGCGCUAUGAUGAAAAGCGGAAUGUUCCUGACCCUGUUCGUGAACACCGUGAUCAACAUUGUGAUCGCCAGCAGGGUGCUGCGGGAGAGGCUGACCGGCAGCCCCUGCGCUGCCUUCAUCGGCGACGACAACAUCGUGAAGGGCGUGAAAAGCGACAAGCUGAUGGCCGACAGGUGCGCCACCUGGCUGAACAUGGAGGUGAAGAUCAUCGACGCCGUGGUGGGCGAGAAGGCCCCCUACUUCUGCGGCGGAUUCAUCCUGUGCGACAGCGUGACCGGCACCGCCUGCAGGGUGGCCGACCCCCUGAAGAGGCUGUUCAAGCUGGGCAAGCCACUGGCCGCUGACGAUGAGCACGACGAUGACAGGCGGAGGGCCCUGCACGAGGAAAGCACCAGGUGGAACAGGGUGGGCAUCCUGAGCGAGCUGUGCAAGGCCGUGGAGAGCAGGUACGAGACCGUGGGCACCAGCAUCAUCGUGAUGGCUAUGACCACACUGGCCAGCUCCGUCAAGAGCUUCUCCUACCUGAGGGGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGGCCGCCACCAUGAGAGUGACAGCCCCUAGAACCUUACUGCUUCUGCUUUGGGGAGCUGUUGCUCUGACAGAGACAUGGGCUGGAUCUUACCACAGCCCCAGCUACGCCUACCACCAGUUCGAGAGGGGGGGAGGAGGCUCCGGGGGAGGAGGCUCCCUGAAGAUCAGCCAGGCCGUGCACGCCGCCCACGCCGAGAUCAACGAGGCCGGCCGGGAGGUGAUCGUGGGCAUUGUCGCUGGCCUGGCCGUCCUCGCCGUGGUGGUGAUUGGAGCUGUGGUCGCAGCUGUUAUGUGCAGAAGAAAGUCAUCCGGCGGAAAGGGAGGCUCCUACUCUCAGGCUGCUUCUGCUACAGUGCCUAGAGCUCUUAUGUGUUUAUCUCAGCUGUAAACUCGAGUAUGUUACGUGCAAAGGUGAUUGUCACCCCCCGAAAGACCAUAUUGUGACACACCCUCAGUAUCACGCCCAAACAUUUACAGCCGCGGUGUCAAAAACCGCGUGGACGUGGUUAACAUCCCUGCUGGGAGGAUCAGCCGUAAUUAUUAUAAUUGGCUUGGUGCUGGCUACUAUUGUGGCCAUGUACGUGCUGACCAACCAGAAACAUAAUUGAAUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA 3067STARR^(TM) 3067 AUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAAAU (SEQgp70- GGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGA ID FLAGGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGG NO:107)UCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCCGUCGACGGCCCCACCAGCCUGUACCACCAGGCCAACAAGGGCGUGAGGGUGGCCUACUGGAUCGGCUUCGACACCACACCCUUCAUGUUCAAGAACCUGGCCGGCGCCUACCCCAGCUACAGCACCAACUGGGCCGACGAGACCGUGCUGACCGCCAGGAACAUCGGCCUGUGCAGCAGCGACGUGAUGGAGAGGAGCCGGAGAGGCAUGAGCAUCCUGAGGAAGAAAUACCUGAAGCCCAGCAACAACGUGCUGUUCAGCGUGGGCAGCACCAUCUACCACGAGAAGAGGGACCUGCUCAGGAGCUGGCACCUGCCCAGCGUGUUCCACCUGAGGGGCAAGCAGAACUACACCUGCAGGUGCGAGACCAUCGUGAGCUGCGACGGCUACGUGGUGAAGAGGAUCGCCAUCAGCCCCGGCCUGUACGGCAAGCCCAGCGGCUACGCCGCUACAAUGCACAGGGAGGGCUUCCUGUGCUGCAAGGUGACCGACACCCUGAACGGCGAGAGGGUGAGCUUCCCCGUGUGCACCUACGUGCCCGCCACCCUGUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGAGCGCCGACGACGCCCAGAAGCUGCUCGUGGGCCUGAACCAGAGGAUCGUGGUCAACGGCAGGACCCAGAGGAACACCAACACAAUGAAGAACUACCUGCUGCCCGUGGUGGCCCAGGCUUUCGCCAGGUGGGCCAAGGAGUACAAGGAGGACCAGGAAGACGAGAGGCCCCUGGGCCUGAGGGACAGGCAGCUGGUGAUGGGCUGCUGCUGGGCCUUCAGGCGGCACAAGAUCACCAGCAUCUACAAGAGGCCCGACACCCAGACCAUCAUCAAGGUGAACAGCGACUUCCACAGCUUCGUGCUGCCCAGGAUCGGCAGCAACACCCUGGAGAUCGGCCUGAGGACCCGGAUCAGGAAGAUGCUGGAGGAACACAAGGAGCCCAGCCCACUGAUCACCGCCGAGGACGUGCAGGAGGCCAAGUGCGCUGCCGACGAGGCCAAGGAGGUGAGGGAGGCCGAGGAACUGAGGGCCGCCCUGCCACCCCUGGCUGCCGACGUGGAGGAACCCACCCUGGAAGCCGACGUGGACCUGAUGCUGCAGGAGGCCGGCGCCGGAAGCGUGGAGACACCCAGGGGCCUGAUCAAGGUGACCAGCUACGACGGCGAGGACAAGAUCGGCAGCUACGCCGUGCUGAGCCCACAGGCCGUGCUGAAGUCCGAGAAGCUGAGCUGCAUCCACCCACUGGCCGAGCAGGUGAUCGUGAUCACCCACAGCGGCAGGAAGGGCAGGUACGCCGUGGAGCCCUACCACGGCAAGGUGGUCGUGCCCGAGGGCCACGCCAUCCCCGUGCAGGACUUCCAGGCCCUGAGCGAGAGCGCCACCAUCGUGUACAACGAGAGGGAGUUCGUGAACAGGUACCUGCACCAUAUCGCCACCCACGGCGGAGCCCUGAACACCGACGAGGAAUACUACAAGACCGUGAAGCCCAGCGAGCACGACGGCGAGUACCUGUACGACAUCGACAGGAAGCAGUGCGUGAAGAAAGAGCUGGUGACCGGCCUGGGACUGACCGGCGAGCUGGUGGACCCACCCUUCCACGAGUUCGCCUACGAGAGCCUGAGGACCAGACCCGCCGCUCCCUACCAGGUGCCCACCAUCGGCGUGUACGGCGUGCCCGGCAGCGGAAAGAGCGGCAUCAUCAAGAGCGCCGUGACCAAGAAAGACCUGGUGGUCAGCGCCAAGAAAGAGAACUGCGCCGAGAUCAUCAGGGACGUGAAGAAGAUGAAAGGCCUGGACGUGAACGCGCGCACCGUGGACAGCGUGCUGCUGAACGGCUGCAAGCACCCCGUGGAGACCCUGUACAUCGACGAGGCCUUCGCUUGCCACGCCGGCACCCUGAGGGCCCUGAUCGCCAUCAUCAGGCCCAAGAAAGCCGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUGAUGUGCCUGAAGGUGCACUUCAACCACGAGAUCUGCACCCAGGUGUUCCACAAGAGCAUCAGCAGGCGGUGCACCAAGAGCGUGACCAGCGUCGUGAGCACCCUGUUCUACGACAAGAAAAUGAGGACCACCAACCCCAAGGAGACCAAAAUCGUGAUCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCUGAUCCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCAGAUCGACUACAAGGGCAACGAGAUCAUGACCGCCGCUGCCAGCCAGGGCCUGACCAGGAAGGGCGUGUACGCCGUGAGGUACAAGGUGAACGAGAACCCACUGUACGCUCCCACCAGCGAGCACGUGAACGUGCUGCUGACCAGGACCGAGGACAGGAUCGUGUGGAAGACCCUGGCCGGCGACCCCUGGAUCAAGACCCUGACCGCCAAGUACCCCGGCAACUUCACCGCCACCAUCGAAGAGUGGCAGGCCGAGCACGACGCCAUCAUGAGGCACAUCCUGGAGAGGCCCGACCCCACCGACGUGUUCCAGAACAAGGCCAACGUGUGCUGGGCCAAGGCCCUGGUGCCCGUGCUGAAGACCGCCGGCAUCGACAUGACCACAGAGCAGUGGAACACCGUGGACUACUUCGAGACCGACAAGGCCCACAGCGCCGAGAUCGUGCUGAACCAGCUGUGCGUGAGGUUCUUCGGCCUGGACCUGGACAGCGGCCUGUUCAGCGCCCCCACCGUGCCACUGAGCAUCAGGAACAACCACUGGGACAACAGCCCCAGCCCAAACAUGUACGGCCUGAACAAGGAGGUGGUCAGGCAGCUGAGCAGGCGGUACCCACAGCUGCCCAGGGCCGUGGCCACCGGCAGGGUGUACGACAUGAACACCGGCACCCUGAGGAACUACGACCCCAGGAUCAACCUGGUGCCCGUGAACAGGCGGCUGCCCCACGCCCUGGUGCUGCACCACAACGAGCACCCACAGAGCGACUUCAGCUCCUUCGUGAGCAAGCUGAAAGGCAGGACCGUGCUGGUCGUGGGCGAGAAGCUGAGCGUGCCCGGCAAGAUGGUGGACUGGCUGAGCGACAGGCCCGAGGCCACCUUCCGGGCCAGGCUGGACCUCGGCAUCCCCGGCGACGUGCCCAAGUACGACAUCAUCUUCGUGAACGUCAGGACCCCAUACAAGUACCACCAUUACCAGCAGUGCGAGGACCACGCCAUCAAGCUGAGCAUGCUGACCAAGAAGGCCUGCCUGCACCUGAACCCCGGAGGCACCUGCGUGAGCAUCGGCUACGGCUACGCCGACAGGGCCAGCGAGAGCAUCAUUGGCGCCAUCGCCAGGCUGUUCAAGUUCAGCAGGGUGUGCAAACCCAAGAGCAGCCUGGAGGAAACCGAGGUGCUGUUCGUGUUCAUCGGCUACGACCGGAAGGCCAGGACCCACAACCCCUACAAGCUGAGCAGCACCCUGACAAACAUCUACACCGGCAGCAGGCUGCACGAGGCCGGCUGCGCCCCCAGCUACCACGUGGUCAGGGGCGAUAUCGCCACCGCCACCGAGGGCGUGAUCAUCAACGCUGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGUGUGCGGCGCCCUGUACAAGAAGUUCCCCGAGAGCUUCGACCUGCAGCCCAUCGAGGUGGGCAAGGCCAGGCUGGUGAAGGGCGCCGCUAAGCACAUCAUCCACGCCGUGGGCCCCAACUUCAACAAGGUGAGCGAGGUGGAAGGCGACAAGCAGCUGGCCGAAGCCUACGAGAGCAUCGCCAAGAUCGUGAACGACAAUAACUACAAGAGCGUGGCCAUCCCACUGCUCAGCACCGGCAUCUUCAGCGGCAACAAGGACAGGCUGACCCAGAGCCUGAACCACCUGCUCACCGCCCUGGACACCACCGAUGCCGACGUGGCCAUCUACUGCAGGGACAAGAAGUGGGAGAUGACCCUGAAGGAGGCCGUGGCCAGGCGGGAGGCCGUGGAAGAGAUCUGCAUCAGCGACGACUCCAGCGUGACCGAGCCCGACGCCGAGCUGGUGAGGGUGCACCCCAAGAGCUCCCUGGCCGGCAGGAAGGGCUACAGCACCAGCGACGGCAAGACCUUCAGCUACCUGGAGGGCACCAAGUUCCACCAGGCCGCUAAGGACAUCGCCGAGAUCAACGCUAUGUGGCCCGUGGCCACCGAGGCCAACGAGCAGGUGUGCAUGUACAUCCUGGGCGAGAGCAUGUCCAGCAUCAGGAGCAAGUGCCCCGUGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCUGCCCUGCCUGUGCAUCCACGCUAUGACACCCGAGAGGGUGCAGCGGCUGAAGGCCAGCAGGCCCGAGCAGAUCACCGUGUGCAGCUCCUUCCCACUGCCCAAGUACAGGAUCACCGGCGUGCAGAAGAUCCAGUGCAGCCAGCCCAUCCUGUUCAGCCCAAAGGUGCCCGCCUACAUCCACCCCAGGAAGUACCUGGUGGAGACCCCACCCGUGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCUGAUCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCAUCAUUAUCGAGGAAGAGGAAGAGGACAGCAUCAGCCUGCUGAGCGACGGCCCCACCCACCAGGUGCUGCAGGUGGAGGCCGACAUCCACGGCCCACCCAGCGUGUCCAGCUCCAGCUGGAGCAUCCCACACGCCAGCGACUUCGACGUGGACAGCCUGAGCAUCCUGGACACCCUGGAGGGCGCCAGCGUGACCUCCGGCGCCACCAGCGCCGAGACCAACAGCUACUUCGCCAAGAGCAUGGAGUUCCUGGCCAGGCCCGUGCCAGCUCCCAGGACCGUGUUCAGGAACCCACCCCACCCAGCUCCCAGGACCAGGACCCCAAGCCUGGCUCCCAGCAGGGCCUGCAGCAGGACCAGCCUGGUGAGCACCCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAACUGGAGGCCCUGACACCCAGCAGGACCCCCAGCAGGUCCGUGAGCAGGACUAGUCUGGUGUCCAACCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAAUUCGAGGCCUUCGUGGCCCAGCAACAGAGACGGUUCGACGCCGGCGCCUACAUCUUCAGCAGCGACACCGGCCAGGGACACCUGCAGCAAAAGAGCGUGAGGCAGACCGUGCUGAGCGAGGUGGUGCUGGAGAGGACCGAGCUGGAAAUCAGCUACGCCCCCAGGCUGGACCAGGAGAAGGAGGAACUGCUCAGGAAGAAACUGCAGCUGAACCCCACCCCAGCCAACAGGAGCAGGUACCAGAGCAGGAAGGUGGAGAACAUGAAGGCCAUCACCGCCAGGCGGAUCCUGCAGGGCCUGGGACACUACCUGAAGGCCGAGGGCAAGGUGGAGUGCUACAGGACCCUGCACCCCGUGCCACUGUACAGCUCCAGCGUGAACAGGGCCUUCUCCAGCCCCAAGGUGGCCGUGGAGGCCUGCAACGCUAUGCUGAAGGAGAACUUCCCCACCGUGGCCAGCUACUGCAUCAUCCCCGAGUACGACGCCUACCUGGACAUGGUGGACGGCGCCAGCUGCUGCCUGGACACCGCCAGCUUCUGCCCCGCCAAGCUGAGGAGCUUCCCCAAGAAACACAGCUACCUGGAGCCCACCAUCAGGAGCGCCGUGCCCAGCGCCAUCCAGAACACCCUGCAGAACGUGCUGGCCGCUGCCACCAAGAGGAACUGCAACGUGACCCAGAUGAGGGAGCUGCCCGUGCUGGACAGCGCUGCCUUCAACGUGGAGUGCUUCAAGAAAUACGCCUGCAACAACGAGUACUGGGAGACCUUCAAGGAGAACCCCAUCAGGCUGACCGAAGAGAACGUGGUGAACUACAUCACCAAGCUGAAGGGCCCCAAGGCCGCUGCCCUGUUCGCUAAGACCCACAACCUGAACAUGCUGCAGGACAUCCCAAUGGACAGGUUCGUGAUGGACCUGAAGAGGGACGUGAAGGUGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGUGCAGGUGAUCCAGGCCGCUGACCCACUGGCCACCGCCUACCUGUGCGGCAUCCACAGGGAGCUGGUGAGGCGGCUGAACGCCGUGCUGCUGCCCAACAUCCACACCCUGUUCGACAUGAGCGCCGAGGACUUCGACGCCAUCAUCGCCGAGCACUUCCAGCCCGGCGACUGCGUGCUGGAGACCGACAUCGCCAGCUUCGACAAGAGCGAGGAUGACGCUAUGGCCCUGACCGCUCUGAUGAUCCUGGAGGACCUGGGCGUGGACGCCGAGCUGCUCACCCUGAUCGAGGCUGCCUUCGGCGAGAUCAGCUCCAUCCACCUGCCCACCAAGACCAAGUUCAAGUUCGGCGCUAUGAUGAAAAGCGGAAUGUUCCUGACCCUGUUCGUGAACACCGUGAUCAACAUUGUGAUCGCCAGCAGGGUGCUGCGGGAGAGGCUGACCGGCAGCCCCUGCGCUGCCUUCAUCGGCGACGACAACAUCGUGAAGGGCGUGAAAAGCGACAAGCUGAUGGCCGACAGGUGCGCCACCUGGCUGAACAUGGAGGUGAAGAUCAUCGACGCCGUGGUGGGCGAGAAGGCCCCCUACUUCUGCGGCGGAUUCAUCCUGUGCGACAGCGUGACCGGCACCGCCUGCAGGGUGGCCGACCCCCUGAAGAGGCUGUUCAAGCUGGGCAAGCCACUGGCCGCUGACGAUGAGCACGACGAUGACAGGCGGAGGGCCCUGCACGAGGAAAGCACCAGGUGGAACAGGGUGGGCAUCCUGAGCGAGCUGUGCAAGGCCGUGGAGAGCAGGUACGAGACCGUGGGCACCAGCAUCAUCGUGAUGGCUAUGACCACACUGGCCAGCUCCGUCAAGAGCUUCUCCUACCUGAGGGGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGGCCGCCACCAUGAGAGUGACAGCCCCUAGAACCUUACUGCUUCUGCUUUGGGGAGCUGUUGCUCUGACAGAGACAUGGGCUGGAUCUCUGAGCGAGGUGACCGGCCAGGGCCUGUGCAUCGGCGCCGUGCCCAAGACCCACCAGGUGCUGUGCAACACCACCCAGAAGACCAGCGACGGCAGCUACUACCUGGCCGCUCCCACCGGCACCACCUGGGCCUGCAGCACCGGCCUGACCCCUUGCAUCAGCACCACCAUCCUGAACCUGACCACCGACUACUGCGUGCUGGUGGAGCUGUGGCCCAGGGUGACCUACCACAGCCCCAGCUACGCCUACCACCAGUUCGAGAGGAGGGCCAAGUACAAGAGGGAGCCCGUGAGCCUGACCCUGGCCCUGCUGCUGGGCGGCCUGACAAUGGGCGGCAUCGCCGCCGGCGUGGGCACCGGCACCACCGCCCUGGUGGCCACCCAGCAGUUCCAGCAGCUGCAGGCCGCCAUGCACGACGACCUGAAGGAGGUGGAGAAGUCCAUCACCAACCUGGAGAAGUCCCUGACCAGCCUGAGCGAGGUGGUGCUGCAGAACAGGAGGGGCCUGGACCUGCUGUUCCUGAAGGAGGGCGGCCUGUGCGCCGCCCUGAAGGAGGAGUGCUGCCUGUACGCCGACCACACCGGCCUGGUGAUCGUGGGCAUUGUCGCUGGCCUGGCCGUCCUCGCCGUGGUGGUGAUUGGAGCUGUGGUCGCAGCUGUUAUGUGCAGAAGAAAGUCAUCCGGCGGAAAGGGAGGCUCCUACUCUCAGGCUGCUUCUGCUACAGUGCCUAGAGCUCUUAUGUGUUUAUCUCAGCUGGGCGGCGGAGGCAGCGACUACAAGGACGACGAUGACAAGUAAACUCGAGUAUGUUACGUGCAAAGGUGAUUGUCACCCCCCGAAAGACCAUAUUGUGACACACCCUCAGUAUCACGCCCAAACAUUUACAGCCGCGGUGUCAAAAACCGCGUGGACGUGGUUAACAUCCCUGCUGGGAGGAUCAGCCGUAAUUAUUAUAAUUGGCUUGGUGCUGGCUACUAUUGUGGCCAUGUACGUGCUGACCAACCAGAAACAUAAUUGAAUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 3068 STARR^(TM) 3068 AUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAAAU (SEQ AH1AGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGA ID 5-GCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGG NO:108) FLAGUCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUGGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCCGUCGACGGCCCCACCAGCCUGUACCACCAGGCCAACAAGGGCGUGAGGGUGGCCUACUGGAUCGGCUUCGACACCACACCCUUCAUGUUCAAGAACCUGGCCGGCGCCUACCCCAGCUACAGCACCAACUGGGCCGACGAGACCGUGCUGACCGCCAGGAACAUCGGCCUGUGCAGCAGCGACGUGAUGGAGAGGAGCCGGAGAGGCAUGAGCAUCCUGAGGAAGAAAUACCUGAAGCCCAGCAACAACGUGCUGUUCAGCGUGGGCAGCACCAUCUACCACGAGAAGAGGGACCUGCUCAGGAGCUGGCACCUGCCCAGCGUGUUCCACCUGAGGGGCAAGCAGAACUACACCUGCAGGUGCGAGACCAUCGUGAGCUGCGACGGCUACGUGGUGAAGAGGAUCGCCAUCAGCCCCGGCCUGUACGGCAAGCCCAGCGGCUACGCCGCUACAAUGCACAGGGAGGGCUUCCUGUGCUGCAAGGUGACCGACACCCUGAACGGCGAGAGGGUGAGCUUCCCCGUGUGCACCUACGUGCCCGCCACCCUGUGCGACCAGAUGACCGGCAUCCUGGCCACCGACGUGAGCGCCGACGACGCCCAGAAGCUGCUCGUGGGCCUGAACCAGAGGAUCGUGGUCAACGGCAGGACCCAGAGGAACACCAACACAAUGAAGAACUACCUGCUGCCCGUGGUGGCCCAGGCUUUCGCCAGGUGGGCCAAGGAGUACAAGGAGGACCAGGAAGACGAGAGGCCCCUGGGCCUGAGGGACAGGCAGCUGGUGAUGGGCUGCUGCUGGGCCUUCAGGCGGCACAAGAUCACCAGCAUCUACAAGAGGCCCGACACCCAGACCAUCAUCAAGGUGAACAGCGACUUCCACAGCUUCGUGCUGCCCAGGAUCGGCAGCAACACCCUGGAGAUCGGCCUGAGGACCCGGAUCAGGAAGAUGCUGGAGGAACACAAGGAGCCCAGCCCACUGAUCACCGCCGAGGACGUGCAGGAGGCCAAGUGCGCUGCCGACGAGGCCAAGGAGGUGAGGGAGGCCGAGGAACUGAGGGCCGCCCUGCCACCCCUGGCUGCCGACGUGGAGGAACCCACCCUGGAAGCCGACGUGGACCUGAUGCUGCAGGAGGCCGGCGCCGGAAGCGUGGAGACACCCAGGGGCCUGAUCAAGGUGACCAGCUACGACGGCGAGGACAAGAUCGGCAGCUACGCCGUGCUGAGCCCACAGGCCGUGCUGAAGUCCGAGAAGCUGAGCUGCAUCCACCCACUGGCCGAGCAGGUGAUCGUGAUCACCCACAGCGGCAGGAAGGGCAGGUACGCCGUGGAGCCCUACCACGGCAAGGUGGUCGUGCCCGAGGGCCACGCCAUCCCCGUGCAGGACUUCCAGGCCCUGAGCGAGAGCGCCACCAUCGUGUACAACGAGAGGGAGUUCGUGAACAGGUACCUGCACCAUAUCGCCACCCACGGCGGAGCCCUGAACACCGACGAGGAAUACUACAAGACCGUGAAGCCCAGCGAGCACGACGGCGAGUACCUGUACGACAUCGACAGGAAGCAGUGCGUGAAGAAAGAGCUGGUGACCGGCCUGGGACUGACCGGCGAGCUGGUGGACCCACCCUUCCACGAGUUCGCCUACGAGAGCCUGAGGACCAGACCCGCCGCUCCCUACCAGGUGCCCACCAUCGGCGUGUACGGCGUGCCCGGCAGCGGAAAGAGCGGCAUCAUCAAGAGCGCCGUGACCAAGAAAGACCUGGUGGUCAGCGCCAAGAAAGAGAACUGCGCCGAGAUCAUCAGGGACGUGAAGAAGAUGAAAGGCCUGGACGUGAACGCGCGCACCGUGGACAGCGUGCUGCUGAACGGCUGCAAGCACCCCGUGGAGACCCUGUACAUCGACGAGGCCUUCGCUUGCCACGCCGGCACCCUGAGGGCCCUGAUCGCCAUCAUCAGGCCCAAGAAAGCCGUGCUGUGCGGCGACCCCAAGCAGUGCGGCUUCUUCAACAUGAUGUGCCUGAAGGUGCACUUCAACCACGAGAUCUGCACCCAGGUGUUCCACAAGAGCAUCAGCAGGCGGUGCACCAAGAGCGUGACCAGCGUCGUGAGCACCCUGUUCUACGACAAGAAAAUGAGGACCACCAACCCCAAGGAGACCAAAAUCGUGAUCGACACCACAGGCAGCACCAAGCCCAAGCAGGACGACCUGAUCCUGACCUGCUUCAGGGGCUGGGUGAAGCAGCUGCAGAUCGACUACAAGGGCAACGAGAUCAUGACCGCCGCUGCCAGCCAGGGCCUGACCAGGAAGGGCGUGUACGCCGUGAGGUACAAGGUGAACGAGAACCCACUGUACGCUCCCACCAGCGAGCACGUGAACGUGCUGCUGACCAGGACCGAGGACAGGAUCGUGUGGAAGACCCUGGCCGGCGACCCCUGGAUCAAGACCCUGACCGCCAAGUACCCCGGCAACUUCACCGCCACCAUCGAAGAGUGGCAGGCCGAGCACGACGCCAUCAUGAGGCACAUCCUGGAGAGGCCCGACCCCACCGACGUGUUCCAGAACAAGGCCAACGUGUGCUGGGCCAAGGCCCUGGUGCCCGUGCUGAAGACCGCCGGCAUCGACAUGACCACAGAGCAGUGGAACACCGUGGACUACUUCGAGACCGACAAGGCCCACAGCGCCGAGAUCGUGCUGAACCAGCUGUGCGUGAGGUUCUUCGGCCUGGACCUGGACAGCGGCCUGUUCAGCGCCCCCACCGUGCCACUGAGCAUCAGGAACAACCACUGGGACAACAGCCCCAGCCCAAACAUGUACGGCCUGAACAAGGAGGUGGUCAGGCAGCUGAGCAGGCGGUACCCACAGCUGCCCAGGGCCGUGGCCACCGGCAGGGUGUACGACAUGAACACCGGCACCCUGAGGAACUACGACCCCAGGAUCAACCUGGUGCCCGUGAACAGGCGGCUGCCCCACGCCCUGGUGCUGCACCACAACGAGCACCCACAGAGCGACUUCAGCUCCUUCGUGAGCAAGCUGAAAGGCAGGACCGUGCUGGUCGUGGGCGAGAAGCUGAGCGUGCCCGGCAAGAUGGUGGACUGGCUGAGCGACAGGCCCGAGGCCACCUUCCGGGCCAGGCUGGACCUCGGCAUCCCCGGCGACGUGCCCAAGUACGACAUCAUCUUCGUGAACGUCAGGACCCCAUACAAGUACCACCAUUACCAGCAGUGCGAGGACCACGCCAUCAAGCUGAGCAUGCUGACCAAGAAGGCCUGCCUGCACCUGAACCCCGGAGGCACCUGCGUGAGCAUCGGCUACGGCUACGCCGACAGGGCCAGCGAGAGCAUCAUUGGCGCCAUCGCCAGGCUGUUCAAGUUCAGCAGGGUGUGCAAACCCAAGAGCAGCCUGGAGGAAACCGAGGUGCUGUUCGUGUUCAUCGGCUACGACCGGAAGGCCAGGACCCACAACCCCUACAAGCUGAGCAGCACCCUGACAAACAUCUACACCGGCAGCAGGCUGCACGAGGCCGGCUGCGCCCCCAGCUACCACGUGGUCAGGGGCGAUAUCGCCACCGCCACCGAGGGCGUGAUCAUCAACGCUGCCAACAGCAAGGGCCAGCCCGGAGGCGGAGUGUGCGGCGCCCUGUACAAGAAGUUCCCCGAGAGCUUCGACCUGCAGCCCAUCGAGGUGGGCAAGGCCAGGCUGGUGAAGGGCGCCGCUAAGCACAUCAUCCACGCCGUGGGCCCCAACUUCAACAAGGUGAGCGAGGUGGAAGGCGACAAGCAGCUGGCCGAAGCCUACGAGAGCAUCGCCAAGAUCGUGAACGACAAUAACUACAAGAGCGUGGCCAUCCCACUGCUCAGCACCGGCAUCUUCAGCGGCAACAAGGACAGGCUGACCCAGAGCCUGAACCACCUGCUCACCGCCCUGGACACCACCGAUGCCGACGUGGCCAUCUACUGCAGGGACAAGAAGUGGGAGAUGACCCUGAAGGAGGCCGUGGCCAGGCGGGAGGCCGUGGAAGAGAUCUGCAUCAGCGACGACUCCAGCGUGACCGAGCCCGACGCCGAGCUGGUGAGGGUGCACCCCAAGAGCUCCCUGGCCGGCAGGAAGGGCUACAGCACCAGCGACGGCAAGACCUUCAGCUACCUGGAGGGCACCAAGUUCCACCAGGCCGCUAAGGACAUCGCCGAGAUCAACGCUAUGUGGCCCGUGGCCACCGAGGCCAACGAGCAGGUGUGCAUGUACAUCCUGGGCGAGAGCAUGUCCAGCAUCAGGAGCAAGUGCCCCGUGGAGGAAAGCGAGGCCAGCACACCACCCAGCACCCUGCCCUGCCUGUGCAUCCACGCUAUGACACCCGAGAGGGUGCAGCGGCUGAAGGCCAGCAGGCCCGAGCAGAUCACCGUGUGCAGCUCCUUCCCACUGCCCAAGUACAGGAUCACCGGCGUGCAGAAGAUCCAGUGCAGCCAGCCCAUCCUGUUCAGCCCAAAGGUGCCCGCCUACAUCCACCCCAGGAAGUACCUGGUGGAGACCCCACCCGUGGACGAGACACCCGAGCCAAGCGCCGAGAACCAGAGCACCGAGGGCACACCCGAGCAGCCACCCCUGAUCACCGAGGACGAGACAAGGACCCGGACCCCAGAGCCCAUCAUUAUCGAGGAAGAGGAAGAGGACAGCAUCAGCCUGCUGAGCGACGGCCCCACCCACCAGGUGCUGCAGGUGGAGGCCGACAUCCACGGCCCACCCAGCGUGUCCAGCUCCAGCUGGAGCAUCCCACACGCCAGCGACUUCGACGUGGACAGCCUGAGCAUCCUGGACACCCUGGAGGGCGCCAGCGUGACCUCCGGCGCCACCAGCGCCGAGACCAACAGCUACUUCGCCAAGAGCAUGGAGUUCCUGGCCAGGCCCGUGCCAGCUCCCAGGACCGUGUUCAGGAACCCACCCCACCCAGCUCCCAGGACCAGGACCCCAAGCCUGGCUCCCAGCAGGGCCUGCAGCAGGACCAGCCUGGUGAGCACCCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAACUGGAGGCCCUGACACCCAGCAGGACCCCCAGCAGGUCCGUGAGCAGGACUAGUCUGGUGUCCAACCCACCCGGCGUGAACAGGGUGAUCACCAGGGAGGAAUUCGAGGCCUUCGUGGCCCAGCAACAGAGACGGUUCGACGCCGGCGCCUACAUCUUCAGCAGCGACACCGGCCAGGGACACCUGCAGCAAAAGAGCGUGAGGCAGACCGUGCUGAGCGAGGUGGUGCUGGAGAGGACCGAGCUGGAAAUCAGCUACGCCCCCAGGCUGGACCAGGAGAAGGAGGAACUGCUCAGGAAGAAACUGCAGCUGAACCCCACCCCAGCCAACAGGAGCAGGUACCAGAGCAGGAAGGUGGAGAACAUGAAGGCCAUCACCGCCAGGCGGAUCCUGCAGGGCCUGGGACACUACCUGAAGGCCGAGGGCAAGGUGGAGUGCUACAGGACCCUGCACCCCGUGCCACUGUACAGCUCCAGCGUGAACAGGGCCUUCUCCAGCCCCAAGGUGGCCGUGGAGGCCUGCAACGCUAUGCUGAAGGAGAACUUCCCCACCGUGGCCAGCUACUGCAUCAUCCCCGAGUACGACGCCUACCUGGACAUGGUGGACGGCGCCAGCUGCUGCCUGGACACCGCCAGCUUCUGCCCCGCCAAGCUGAGGAGCUUCCCCAAGAAACACAGCUACCUGGAGCCCACCAUCAGGAGCGCCGUGCCCAGCGCCAUCCAGAACACCCUGCAGAACGUGCUGGCCGCUGCCACCAAGAGGAACUGCAACGUGACCCAGAUGAGGGAGCUGCCCGUGCUGGACAGCGCUGCCUUCAACGUGGAGUGCUUCAAGAAAUACGCCUGCAACAACGAGUACUGGGAGACCUUCAAGGAGAACCCCAUCAGGCUGACCGAAGAGAACGUGGUGAACUACAUCACCAAGCUGAAGGGCCCCAAGGCCGCUGCCCUGUUCGCUAAGACCCACAACCUGAACAUGCUGCAGGACAUCCCAAUGGACAGGUUCGUGAUGGACCUGAAGAGGGACGUGAAGGUGACACCCGGCACCAAGCACACCGAGGAGAGGCCCAAGGUGCAGGUGAUCCAGGCCGCUGACCCACUGGCCACCGCCUACCUGUGCGGCAUCCACAGGGAGCUGGUGAGGCGGCUGAACGCCGUGCUGCUGCCCAACAUCCACACCCUGUUCGACAUGAGCGCCGAGGACUUCGACGCCAUCAUCGCCGAGCACUUCCAGCCCGGCGACUGCGUGCUGGAGACCGACAUCGCCAGCUUCGACAAGAGCGAGGAUGACGCUAUGGCCCUGACCGCUCUGAUGAUCCUGGAGGACCUGGGCGUGGACGCCGAGCUGCUCACCCUGAUCGAGGCUGCCUUCGGCGAGAUCAGCUCCAUCCACCUGCCCACCAAGACCAAGUUCAAGUUCGGCGCUAUGAUGAAAAGCGGAAUGUUCCUGACCCUGUUCGUGAACACCGUGAUCAACAUUGUGAUCGCCAGCAGGGUGCUGCGGGAGAGGCUGACCGGCAGCCCCUGCGCUGCCUUCAUCGGCGACGACAACAUCGUGAAGGGCGUGAAAAGCGACAAGCUGAUGGCCGACAGGUGCGCCACCUGGCUGAACAUGGAGGUGAAGAUCAUCGACGCCGUGGUGGGCGAGAAGGCCCCCUACUUCUGCGGCGGAUUCAUCCUGUGCGACAGCGUGACCGGCACCGCCUGCAGGGUGGCCGACCCCCUGAAGAGGCUGUUCAAGCUGGGCAAGCCACUGGCCGCUGACGAUGAGCACGACGAUGACAGGCGGAGGGCCCUGCACGAGGAAAGCACCAGGUGGAACAGGGUGGGCAUCCUGAGCGAGCUGUGCAAGGCCGUGGAGAGCAGGUACGAGACCGUGGGCACCAGCAUCAUCGUGAUGGCUAUGACCACACUGGCCAGCUCCGUCAAGAGCUUCUCCUACCUGAGGGGGGCCCCUAUAACUCUCUACGGCUAACCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGGCCGCCACCAUGAGAGUGACAGCCCCUAGAACCUUACUGCUUCUGCUUUGGGGAGCUGUUGCUCUGACAGAGACAUGGGCUGGAUCUUACCACAGCCCCAGCUACGCCUACCACCAGUUCGAGAGGGGGGGAGGAGGCUCCGGGGGAGGAGGCUCCCUGAAGAUCAGCCAGGCCGUGCACGCCGCCCACGCCGAGAUCAACGAGGCCGGCCGGGAGGUGAUCGUGGGCAUUGUCGCUGGCCUGGCCGUCCUCGCCGUGGUGGUGAUUGGAGCUGUGGUCGCAGCUGUUAUGUGCAGAAGAAAGUCAUCCGGCGGAAAGGGAGGCUCCUACUCUCAGGCUGCUUCUGCUACAGUGCCUAGAGCUCUUAUGUGUUUAUCUCAGCUGGGCGGCGGAGGCAGCGACUACAAGGACGACGAUGACAAGUAAACUCGAGUAUGUUACGUGCAAAGGUGAUUGUCACCCCCCGAAAGACCAUAUUGUGACACACCCUCAGUAUCACGCCCAAACAUUUACAGCCGCGGUGUCAAAAACCGCGUGGACGUGGUUAACAUCCCUGCUGGGAGGAUCAGCCGUAAUUAUUAUAAUUGGCUUGGUGCUGGCUACUAUUGUGGCCAUGUACGUGCUGACCAACCAGAAACAUAAUUGAAUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUCAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA non structural protein of SINVmARM # 2842 SINV MEKPVVNVDVDPQSPFVVQLQKSFPQFEVVAQQVTPNDHANARAFS andnsP1- HLASKLIELEVPTTATILDIGSAPARRMFSEHQYHCVCPMRSPEDP 2862 4 AADRMMKYASKLAEKACKITNKNLHEKIKDLRTVLDTPDAETPSLCFH (SEQNDVTCNMRAEYSVMQDVYINAPGTIYHQAMKGVRTLYWIGFDTTQF IDMFSAMAGSYPAYNTNWADEKVLEARNIGLCSTKLSEGRTGKLSIMR NO:109)KKELKPGSRVYFSVGSTLYPEHRASLQSWHLPSVFHLNGKQSYTCRCDTVVSCEGYVVKKITISPGITGETVGYAVTHNSEGFLLCKVTDTVKGERVSFPVCTYIPATICDQMTGIMATDISPDDAQKLLVGLNQRIVINGRTNRNTNTMQNYLLPIIAQGFSKWAKERKDDLDNEKMLGTRERKLTYGCLWAFRTKKVHSFYRPPGTQTCVKVPASFSAFPMSSVWTTSLPMSLRQKLKLALQPKKEEKLLQVSEELVMEAKAAFEDAQEEARAEKLREALPPLVADKGIEAAAEVVCEVEGLQADIGAALVETPRGHVRIIPQANDRMIGQYIVVSPNSVLKNAKLAPAHPLADQVKIITHSGRSGRYAVEPYDAKVLMPAGGAVPWPEFLALSESATLVYNEREFVNRKLYHIAMHGPAKNTEEEQYKVTKAELAETEYVFDVDKKRCVKKEEASGLVLSGELTNPPYHELALEGLKTRPAVPYKVETIGVIGTPGSGKSAIIKSTVTARDLVTSGKKENCREIEADVLRLRGMQITSKTVDSVMLNGCHKAVEVLYVDEAFACHAGALLALIAIVRPRKKVVLCGDPMQCGFFNMMQLKVHFNHPEKDICTKTFYKYISRRCTQPVTAIVSTLHYDGKMKTTNPCKKNIEIDITGATKPKPGDIILTCFRGWVKQLQIDYPGHEVMTAAASQGLTRKGVYAVRQKVNENPLYAITSEHVNVLLTRTEDRLVWKTLQGDPWIKQLTNIPKGNFQATIEDWEAEHKGIIAAINSPTPRANPFSCKTNVCWAKALEPILATAGIVLTGCQWSELFPQFADDKPHSAIYALDVICIKFFGMDLTSGLFSKQSIPLTYHPADSARPVAHWDNSPGTRKYGYDHAIAAELSRRFPVFQLAGKGTQLDLQTGRTRVISAQHNLVPVNRNLPHALVPEYKEKQPGPVEKFLNQFKHHSVLVVSEEKIEAPRKRIEWIAPIGIAGADKNYNLAFGFPPQARYDLVFINIGTKYRNHHFQQCEDHAATLKTLSRSALNCLNPGGTLVVKSYGYADRNSEDVVTALARKFVRVSAARPDCVSSNTEMYLIFRQLDNSRTRQFTPHHLNCVISSVYEGTRDGVGAAPSYRTKRENIADCQEEAVVNAANPLGRPGEGVCRAIYKRWPTSFTDSATETGTARMTVCLGKKVIHAVGPDFRKHPEAEALKLLQNAYHAVADLVNEHNIKSVAIPLLSTGIYAAGKDRLEVSLNCLTTALDRTDADVTIYCLDKKWKERIDAALQLKESVTELKDEDMEIDDELVWIHPDSCLKGRKGFSTTKGKLYSYFEGTKFHQAAKDMAEIKVLFPNDQESNEQLCAYILGETMEAIREKCPVDHNPSSSPPKTLPCLCMYAMTPERVHRLRSNNVKEVTVCSSTPLPKHKIKNVQKVQCTKVVLFNPHTPAFVPARKYIEVPEQPTAPPAQAEEAPEVVATPSPSTADNTSLDVTDISLDMDDSSEGSLFSSFSGSDNSITSMDSWSSGPSSLEIVDRRQVVVADVHAVQEPAPIPPPRLKKMARLAAARKEPTPPASNSSESLHLSFGGVSMSLGSIFDGETARQAAVQPLATGPTDVPMSFGSFSDGEIDELSRRVTESEPVLFGSFEPGEVNSIISSRSAVSFPLRKQRRRRRSRRTEY*LTGVGGYIFSTDTGPGHLQKKSVLQNQLTEPTLERNVLERIHAPVLDTSKEEQLKLRYQMMPTEANKSRYQSRKVENQKAITTERLLSGLRLYNSATDQPECYKITYPKPLYSSSVPANYSDPQFAVAVCNNYLHENYPTVASYQITDEYDAYLDMVDGTVACLDTATFCPAKLRSYPKKHEYRAPNIRSAVPSAMQNTLQNVLIAATKRNCNVTQMRELPTLDSATFNVECFRKYACNDEYWEEFARKPIRITTEFVTAYVARLKGPKAAALFAKTYNLVPLQEVPMDRFVMDMKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYICGIHRELVRRLTAVLLPNIHTLFDMSAEDFDAIIAEHFKQGDPVLETDIASFDKSQDDAMALTGLMILEDLGVDQPLLDLIECAFGEISSTHLPTGTRFKFGAMMKSGMFLTLFVNTVLNVVIASRVLEERLKTSRCAAFIGDDNIIHGVVSDKEMAERCATWLNMEVKIIDAVIGERPPYFCGGFILQDSVTSTACRVADPLKRLFKLGKPLPADDEQDEDRRRALLDETKAWFRVGITGTLAVAVTTRYEVDNITPVLLALRTFAQSKRAFQAIRGEIKHLYGGPK

Example 11

This example describes analysis of the immunogenicity of influenzahemagglutinin (HA) expressed from self-replicating RNA or mRNA.

Self-replicating RNA and mRNA vaccine constructs were designed to encodethe full-length hemagglutinin (HA) protein from influenza virusA/California/07/2009 (H1N1) (SEQ ID NO:113 and 114). As described abovefor Example 1, the mRNA vaccine construct encoding HA included a tobaccoetch virus (TEV) 5′ UTR and a Xenopus beta-globin (Xbg) 3′ UTR. Bothself-replicating RNA (SEQ ID NO:56; entire RNA mARM3039) and mRNAvaccine constructs (SEQ ID NO: 116; entire RNA sequence mARM3038) wereencapsulated in the same lipid nanoparticle (LNP) composition thatincluded four lipid excipients (an ionizable cationic lipid,1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, andPEG2000-DMG) dispersed in HEPES buffer (pH 8.0) containing sodiumchloride and the cryoprotectants sucrose and glycerol. The N:P ratio ofcomplexing lipid and RNA was approximately 9:1. The ionizable cationiclipid had the following structure:

Five female, 8-10 week old Balb/c mice were injected intramuscularlywith 2 mg of mRNA or self-replicating RNA encoding HA. Mice were bled ondays 14, 28, 42, and 56, followed by hemagglutination inhibition (HAI)assay using serially diluted sera. The reciprocal of the highestdilution of serum that caused inhibition of hemagglutination wasconsidered the HAI titer, with a titer of 1/40 being protective againstinfluenza virus infection and four-fold higher titers than baselineindicating seroconversion.

Results in FIG. 23 show that greater HAI titers were obtained withself-replicating RNA encoding HA as compared to mRNA encoding HA. HAItiters for the self-replicating RNA construct encoding HA were greaterthan HAI titers for the mRNA encoding HA at all time points beginning atday 28. In addition, protective HAI titers were seen for theself-replicating RNA construct encoding HA beginning at day 28 that weremaintained for at least 56 days. By contrast, mRNA encoding HA showedprotective HAI titers only at day 56 that were lower than HAI titersseen for the self-replicating RNA HA construct. At all other timepoints, HAI titers for the mRNA construct encoding HA were below theprotective titer threshold, with an HAI titer that was comparable toinjection with PBS control at day 28.

These results show that the self-replicating RNA construct encoding HAelicited protective HA antibody titers, with greater HAI titers ascompared to the mRNA construct encoding HA.

Example 12

This example describes dsRNA production and luciferase expression forself-replicating RNA.

Several self-replicating RNA systems from different alphaviruses weretested for expression in vitro using either green fluorescent protein(GFP) or firefly luciferase (Luc) as reporter genes. Initialtransfection of cells with increasing amounts of self-replicating RNAresulted in expression of reporter genes at a lower dose compared tomRNA. However, as the amount of input self-replicating RNA increased,detectable expression of the reporter gene decreased.

Self-replicating RNA produces double stranded RNA (dsRNA) as anintermediate in the amplification process. Overproduction of dsRNA cansuppress translation. To evaluate the effect of dsRNA production ontransgene expression, dsRNA and the expression of reporter geneluciferase were measured simultaneously. HEK293 cells were transfectedwith 2 μg of replicon A (SEQ ID NO:115; entire RNA sequence mARM2826) orreplicon B (SEQ ID NO:100, entire RNA sequence mARM2809)self-replicating RNA, or mRNA expressing Luc (SEQ ID NO:102, entire mRNAsequence mARM1782) using a commercial RNA transfection reagent.Untransfected cells (UTC) served as a control. dsRNA production (FIG.24A) was quantified using immunohistochemical staining for dsRNA,followed by fluorescence quantification using a fluorescence scanner 24hours after transfection. Luciferase expression (FIG. 24B) was assayedby measuring bioluminescence in parallel.

Replicon A produced a 3-fold higher level of dsRNA than replicon B 24hrs after transfection (FIG. 24A). However, replicon B produced a2.4-fold higher expression level of luciferase compared to replicon A.Furthermore, the level of luciferase expression from replicon A wasequivalent to that observed for mRNA. Thus, even though replicon A hadthe ability to amplify the amount of replicon RNA and transcribed mRNAencoding luciferase, translation of the amplified mRNA was inhibited,consistent with overproduction of dsRNA inhibiting translation.Furthermore, higher levels of luciferase gene expression were seen forreplicon RNA as compared to mRNA at 24, 48, and 72 hours aftertransfection of HEK293 cells (FIG. 15A). Self-replicating RNA with anexpression cassette that included a luciferase reporter gene followed byan IRES and E3L also showed robust luciferase expression (FIGS. 15B,15C; SEQ ID NOs: 128 and 129). Luciferase expression was also seen for aself-replicating RNA that expressed E3L from a first subgenomic promoterand a luciferase reporter gene from a second subgenomic promoter located3′ of the E3L open reading frame (not shown). Thus, not only didreplicon RNA produce higher levels of luciferase gene expressioncompared to mRNA, but replicon RNA also showed increased duration ofexpression over a 72-hr period.

Example 13

This example describes immunogenicity of liquid and lyophilizedself-replicating RNA formulations. Immunogenicity of self-replicatingRNA (SEQ ID NO:125) formulated as a lyophilized lipid nanoparticle(LYO-LNP) was tested in BALB/c mice in two separate preclinical studiesand compared with the liquid (frozen) LNP formulation (Liquid-LNP). Eachstudy included the use of a PBS dosing group as a negative control and aLiquid dosing group (Liquid-LNP) as a positive control. Both LYO-LNP andLiquid-LNP formulations were dosed at 0.2 and 2 μg. There were n=5animals per dose group in each study. Test formulations wereadministered intramuscularly (IM) and serum was collected at varioustimepoints (Days 10, 19, 31 for the first study and Days 10, 20, 30 forthe second study) post-immunization to measure the production ofanti-SARS-CoV-2 spike protein IgG using a Luminex bead fluorescentassay.

In both studies, anti-SARS-CoV-2 spike protein IgGs were detected inserum in a time- and dose-dependent manner for both Liquid-LNP andLYO-LNP formulations, whereas PBS injection did not elicit animmunogenic response (FIG. 16A-16D). There was no statistical differencein immunogenicity seen between Liquid-LNP and LYO-LNP dose groups in thefirst study, whereas LYO-LNP produced statistically different andgreater IgG than Liquid-LNP in the second study. Without being limitedby theory, under-powering (n=5/group) of these two separate studies mayhave contributed to the statistical differences in immunogenicityresults observed in the two studies. In combining the results of bothstudies, no statistically significant differences were observed betweenLiquid-LNP and LYO-LNP formulations at the 0.2 and 2 μg dose levels(FIG. 17A, 17B). Taken together, the results of these studiesdemonstrate that the immunogenicity of the liquid and lyophilizedformulations were comparable.

In summary, the liquid and lyophilized formulations of theself-replicating RNA vaccine (SEQ ID NO:125) showed comparableimmunogenicity. The vaccine can induce effective, adaptive humoral(neutralizing antibodies) and cellular (CD8+) immune responses targetingthe SARS-CoV-2 S glycoprotein. The vaccine also elicits induction ofanti-spike glycoprotein antibodies (IgG) levels that are higher than aconventional mRNA vaccine and also induces production of IgG antibodiesat a faster rate than a conventional mRNA vaccine. It continues toproduce increasing levels of IgG up to 50 days post vaccination whereasthe conventional mRNA vaccine plateaus by day 10 post vaccination. Itproduces an RNA dose-dependent increase in CD8+ T lymphocytes and abalanced, Th1 dominant CD4+ T helper cell immune response with no skewtowards a Th2 response.

Any and all references and citations to other documents, such aspatents, patent applications, patent publications, journals, books,papers, web contents, that have been made throughout this disclosure arehereby incorporated herein in their entirety for all purposes.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

Example 14

Lyophilization of Self-Replicating RNA-Lipid Nanoparticle FormulationMaterials and Methods Generally

The processes conducted in this example were conducted using lipidnanoparticle compositions that were manufactured according to well-knownprocesses, for example, those described in U.S. application Ser. No.16/823,212, the contents of which are incorporated by reference for thespecific purpose of teaching lipid nanoparticle manufacturing processes.The lipid nanoparticle compositions and the lyophilized products werecharacterized for several properties. The materials and methods forthese characterization processes as well as a general method ofmanufacturing the lipid nanoparticle compositions that were used forlyophilization experiments are provided in this example.

Lipid Nanoparticle Manufacture

Lipid nanoparticle formulations used in this example were manufacturedby mixing lipids (ionizable cationic lipid (ATX-126):helperlipid:cholesterol:PEG-lipid) in ethanol with RNA dissolved in citratebuffer. The mixed material was instantaneously diluted with PhosphateBuffer. Ethanol was removed by dialysis against phosphate buffer usingregenerated cellulose membrane (100 kD MWCO) or by tangential flowfiltration (TFF) using modified polyethersulfone (mPES) hollow fibermembranes (100 kD MWCO). Once the ethanol was completely removed, thebuffer was exchanged with HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer containing10-300 (for example, 40-60) mM NaCl and 5-15% sucrose, pH 7.3. Theformulation was concentrated followed by 0.2 μm filtration using PESfilters. The RNA concentration in the formulation was then measured byRiboGreen fluorimetric assay, and the concentration was adjusted to afinal desired concentration by diluting with HEPES buffer containing10-100 (for example 40-60) mM NaCl, 0-15% sucrose, pH 7.2-8.5 containingglycerol. If not used immediately for further studies, the finalformulation was then filtered through a 0.2 μm filter and filled intoglass vials, stoppered, capped and placed at −70 t 5° C. The lipidnanoparticles formulations were characterized for their pH andosmolality. Lipid Content and RNA content were measured by highperformance liquid chromatography (HPLC), and mRNA integrity by wasmeasured by fragment analyzer.

Dynamic Light Scattering (DLS)

The average particle size (z) and polydispersity index (PD1) of lipidnanoparticle formulations used in the Examples was measured by dynamiclight scattering on a Malvern Zetasizer Nano ZS (United Kingdom).

RiboGreen Assay

The encapsulation efficiency of the lipid nanoparticle formulations wascharacterized using the RiboGreen fluorometric assay. RiboGreen is aproprietary fluorescent dye (Molecular Probes/Invitrogen a division ofLife Technologies, now part of Thermo Fisher Scientific of Eugene,Oreg., United States) that is used in the detection and quantificationof nucleic acids, including both RNA and DNA. In its free form,RiboGreen exhibits little fluorescence and possesses a negligibleabsorbance signature. When bound to nucleic acids, the dye fluoresceswith an intensity that is several orders of magnitude greater than theunbound form. The fluorescence can be then be detected by a sensor(fluorimeter) and the nucleic acid can be quantified.

Lyophilization Process

Self-Replicating RNAs (aka Replicon RNA) are typically larger than theaverage mRNA, and tests were designed to determine whetherself-replicating RNA lipid nanoparticle formulations could besuccessfully lyophilized. The quality of lyophilized lipid nanoparticleformulations was assessed by analyzing the formulationspost-lyophilization and comparing this to the lipid nanoparticleformulation prior to lyophilization as well as after a conventionalfreeze/thaw cycle (i.e., frozen at ˜−70° C. then allowed to thaw at roomtemperature).

The analysis of the lipid nanoparticle formulations included theanalysis of particle size and polydispersity (PD1) and encapsulationefficiency (% Encap). The particle size post-lyophilization was comparedto the particle size pre-lyophilization and the difference can bereported as a delta (8). The various compositions tested were screenedas to whether a threshold of properties was met including minimalparticle size increase (8<10 nm), the maintenance of PD1 (<0.2), andmaintenance of high encapsulation efficiency (>85%).

The lipid nanoparticle formulations were prepared as described above,with self-replicating RNA (SEQ ID NO: 125). The resulting lipidnanoparticle formulation was then processed with a buffer exchange toform a prelyophilization suspension having a concentration of 0.05 to2.0 mg/mL self-replicating RNA, 0.01 to 0.05 M potassium sorbate, 0.01to 0.10% w/v Poloxamer 188 (Kolliphor®), 14 to 18% w/v sucrose, 25 to 75mM NaCl, and 15 to 25 mM pH 8.0 Tris buffer. The prelyophilizationformulation was then lyophilized in a Millrock Revo Freeze Dryer (ModelNo. RV85S4), using aliquots of 2.0 mL of suspension and thelyophilization cycle provided in Table 10 below. The lyophilizedformulations of this example were then applied to the studies of Example13 above as “LYO-LNP”.

TABLE 10 Lyophilization Cycle for Self-Replicating RNA-LipidNanoparticle Formulation Freeze drying cycle shelf step chambertemperature duration vacuum Step (° C., ±2° C.) (h:min) (mbar) InitialFreezing −50 4:00 atmosphere Evacuation −50 00:30-01:45 from atmosph.pressure to 0.05 Primary drying −50 → 0 63:00 0.05 (ramp down) Secondarydrying 0 → +25 39:30 0.05 (ramp up) Backfill with N₂ and 25 00:10-00:20700 ± 50 stoppering Aeration with air 5 00:10-00:20 atmosphere

The lyophilized particles prepared following the methods described abovewere reconstituted in 2 mL of water and characterized using DLS andRiboGreen. The results provided in Table 11 below show that thelyophilized compositions were found to produce lyophilized lipidnanoparticle formulations with adequate size, polydispersity, and deltavalues (˜5.3 nm) upon reconstitution.

TABLE 11 Self-Replicating RNA-Lipid Nanoparticle Characteristics Pre-and Post-LYO Average Particle Size (nm) PDI encap (%) Pre-LYO 76.3 0.12997 Post-LYO 81.6 0.152 93

What is claimed is:
 1. A nucleic acid molecule comprising (a) a sequenceof SEQ ID NO:124; (b) a sequence of SEQ ID NO:124, wherein T issubstituted with U; (c) a sequence of SEQ ID NO:125; or (d) a sequenceof SEQ ID NO:125, wherein T is substituted with U.
 2. A compositioncomprising: (I) a nucleic acid molecule comprising: (A) a firstpolynucleotide encoding one or more viral replication proteins, whereinthe first polynucleotide is codon-optimized as compared to a wild-typepolynucleotide encoding the one or more viral replication proteins; and(B) a second polynucleotide comprising a transgene encoding an antigenicprotein or a fragment thereof, wherein the antigenic protein is acoronavirus protein; and (II) a lipid formulation selected from alipoplex, a liposome, a lipid nanoparticle, a polymer-based carrier, anexosome, a lamellar body, a micelle, and an emulsion, wherein thenucleic acid molecule comprises (a) a sequence of SEQ ID NO:124; (b) asequence of SEQ ID NO:124, wherein T is substituted with U; (c) asequence of SEQ ID NO:125; or (d) a sequence of SEQ ID NO:125, wherein Tis substituted with U.
 3. The composition of claim 2, wherein the lipidformulation is a lipid nanoparticle having a size of less than about 200nm.
 4. The composition of claim 2, wherein the lipid formulationcomprises an ionizable cationic lipid.
 5. The composition of claim 4,wherein the ionizable cationic lipid has a structure of Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein R⁵ andR⁶ are each independently selected from the group consisting of a linearor branched C₁-C₃₁ alkyl, C₂-C₃₁ alkenyl or C₂-C₃₁ alkynyl andcholesteryl; L⁵ and L⁶ are each independently selected from the groupconsisting of a linear C₁-C₂₀ alkyl and C₂-C₂₀ alkenyl; X⁵ is —C(O)O—,whereby —C(O)O—R⁶ is formed or —OC(O)— whereby —OC(O)—R⁶ is formed; X⁶is —C(O)O— whereby —C(O)O—R⁵ is formed or —OC(O)— whereby —OC(O)—R⁵ isformed; X⁷ is S or O; L⁷ is absent or lower alkyl; R⁴ is a linear orbranched C₁-C₆ alkyl; and R⁷ and R⁸ are each independently selected fromthe group consisting of a hydrogen and a linear or branched C₁-C₆ alkyl.6. The composition of claim 4, wherein the ionizable cationic lipid isATX-126:


7. The composition of claim 2, wherein the lipid formulationencapsulates the nucleic acid molecule or is complexed to the nucleicacid molecule.
 8. The composition of claim 2, wherein the lipidformulation comprises (i) a helper lipid; (ii) a phospholipid; (iii) apolyethylene glycol (PEG)-lipid conjugate; or (iv) any combinationthereof.
 9. The composition of claim 8, wherein the phospholipid isselected from dioleoylphosphatidyl ethanolamine (DOPE),dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidyl choline(DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), and phosphatidylcholine (PC).
 10. Thecomposition of claim 8, wherein the PEG-lipid conjugate is PEG-DMG. 11.The composition of claim 2, wherein the lipid portion of the lipidformulation comprises about 40 mol % to about 60 mol % of the ionizablecationic lipid, about 4 mol % to about 16 mol % DSPC, about 30 mol % toabout 47 mol % cholesterol, and about 0.5 mol % to about 3 mol %PEG2000-DMG.
 12. The composition of claim 2, wherein the composition hasa total lipid:nucleic acid molecule weight ratio of about 50:1 to about10:1.
 13. The composition of claim 2, wherein the composition furthercomprises (i) a HEPES or TRIS buffer at a pH of about 7.0 to about 8.5;(ii) a HEPES or TRIS buffer at a concentration of about 7 mg/mL to about15 mg/mL; (iii) about 2.0 mg/mL to about 4.0 mg/mL of NaCl; (iv) one ormore cryoprotectants; (v) one or more cryoprotectants selected fromsucrose, glycerol, or a combination of sucrose and glycerol; or (vi) anycombination thereof.
 14. The composition of claim 13, wherein thecomposition comprises a combination of sucrose at a concentration ofabout 70 mg/mL to about 110 mg/mL and glycerol at a concentration ofabout 50 mg/mL to about 70 mg/mL.
 15. The composition of claim 2,wherein the composition is a lyophilized composition.
 16. Thecomposition of claim 15, wherein the lyophilized composition comprisesone or more lyoprotectants.
 17. The composition of claim 15, wherein thelyophilized composition comprises a poloxamer, potassium sorbate,sucrose, or any combination thereof.
 18. The composition of claim 15,wherein the lyophilized composition comprises (i) about 0.01 to about1.0% w/w of the nucleic acid molecule; (ii) about 1.0 to about 5.0% w/wlipids; (iii) about 0.5 to about 2.5% w/w of TRIS buffer; (iv) about0.75 to about 2.75% w/w of NaCl; (v) about 85 to about 95% w/w of asugar; (vi) about 0.01 to about 1.0% w/w of a poloxamer; (vii) about 1.0to about 5.0% w/w of potassium sorbate; or (viii) any combinationthereof.
 19. The composition of claim 18, wherein the sugar is sucrose.20. The composition of claim 18, wherein the poloxamer is poloxamer 188.21. A lipid nanoparticle composition comprising (a) a lipid formulationcomprising (i) about 45 mol % to about 55 mol % of an ionizable cationiclipid having the structure of ATX-126:

(ii) about 8 mol % to about 12 mol % DSPC; (iii) about 35 mol % to about42 mol % cholesterol; and (iv) about 1.25 mol % to about 1.75 mol %PEG2000-DMG; and (b) a nucleic acid molecule having at least 85%sequence identity to SEQ ID NO:125; wherein the lipid formulationencapsulates the nucleic acid molecule and the lipid nanoparticle has asize of about 60 to about 90 nm.
 22. A method of administering thecomposition of claim 2 to a subject in need thereof, wherein thecomposition is lyophilized and is reconstituted prior to administration.23. A method of preventing or ameliorating COVID-19, comprisingadministering the composition of claim 2 to a subject in need thereof.24. The method of claim 23, wherein the composition is administered onetime or two times.
 25. A method of administering a booster dose to avaccinated subject, comprising administering the composition of claim 2to a subject who was previously vaccinated against coronavirus.
 26. Themethod of claim 23, wherein the composition is administered at a dosageof about 0.01 μg to about 1,000 μg of nucleic acid.
 27. A method ofinducing an immune response against a coronavirus in a subjectcomprising: administering to the subject an effective amount of anucleic acid molecule of claim
 1. 28. A method of inducing an immuneresponse against a coronavirus in a subject comprising: administering tothe subject an effective amount of a composition of claim 2.